Graphical abstract
Highlights
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Graphene oxide-coated titanate on the surface of Ti implants was prepared.
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The structure was excellent vehicles for dexamethasone delivery.
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The structure with dexamethasone promoted cell proliferation and osteo-differentiation.
Abstract
Objective
In this study, the objective is to construct graphene-oxide-coated titanate on titanium foils as drug vehicle to enhance cell proliferation and osteo-differentiation of rat bone mesenchymal stem cells (rBMSCs).
Methods
Graphene oxide (GO) sheets obtained using the modified Hummer’s method and characterized by AFM were coupled with bioactive titanate on Ti implants (GO-Ti) pretreated by alkali, followed by reduction (rGO-Ti). They were characterized by Raman spectroscopy, XPS, SEM, FTIR and contact angle. After dexamethasone (DEX) was loaded onto them (DEX-GO-Ti and DEX-rGO-Ti), cell proliferation of rBMSCs on them was evaluated by CCK-8 and F-actin staining, and differentiation through alkaline phosphatase activity, mRNA expression, and calcium nodules.
Results
The obtained GO sheets were monolayers from AFM. Raman spectra exhibited two prominent peaks at D and G bands, and the I(D)/I(G) ratios increased from 0.96 to 1.68 after reduction. XPS proved the existence of oxygenated functional groups for GO-Ti and the reduction of their intensity for rGO-Ti. From SEM, GO and rGO were evenly coated on nanostructures. DEX-GO-Ti absorbed most amount of DEX and released in a sustained manner. CCK-8 results showed that DEX-GO-Ti showed excellent performance on promoting cell proliferation. RMBSCs on DEX-GO-Ti presented greatly high expression of calcium, proteins and mRNA related to osteogenic differentiation.
Significance
GO coated titanate nanostructrues on surfaces of Ti foils by a simple self-assembly method, showed excellent vechiles for DEX. The construct promoted proliferation and accelerated osteogenic differentiation of rBMSCs, and would be prosperous for their further applications.
1
Introduction
Titanium (Ti)-based materials have demonstrated their superiority among metallic materials in the biomedical field, such as orthopedics and dentistry, due to their excellent mechanical strength and favorable biocompatibility . However, there are some disadvantages of current Ti-based implant materials that may hinder their further applications. For example, the biological inertness of Ti may induce the generation of fibrous tissue, which causes micro-movement between the bone and the interface, and ultimately, leads to the failure of implants. Various trials have been proposed to address this issue, and one of the current methods is to spontaneously deposit on the surface hydroxyapatite, similar to the composition of mineral part of the bone or osteoinductive ions . The hydroxyapatite coating has been shown to greatly improve the bioactivity of implants and accelerate osteogenic differentiation, but the poor mechanical properties and its weak bonding to the substrate usually affect the long-term reliability under load .
In the past few years, graphene, a flat monolayer of carbon atoms closely packed into a honeycomb shaped two-dimensional lattice, has attracted increasing attentions for its wide applications in biomedicine, such as drug delivery carriers, imaging agents and tissue engineering substrates due to their outstanding physicochemical, optical and mechanical properties . Being only one atom thick, it introduces the least amount of artificial material possible and has a large number of remarkable properties, which can confer beneficial properties on implants to minimize the possibility of inflammatory responses caused by the implantation of foreign materials . Graphene has the highest Young’s modulus (0.5–1 TPa) among any known material and is not brittle ; the excellent mechanical properties endow it to play a key role in hard tissue engineering . The derivative, graphene oxide (GO) consists of hydrophobic π domains in the core region and ionized groups around the GO edges. These are features that substantially enhance its interactions with proteins through hydrophobic and electrostatic interactions and they could potentially enhance the specific differentiation of mesenchymal stem cells . Evidences have shown that GO promoted the adhesion, proliferation and mineralization in the presence of osteogenic chemical inducers . It was reported that the strong noncovalent binding abilities of graphene allowed it to act as a preconcentration platform for osteogenic inducers, which further accelerated osteogenic differentitation .
Based on these advantages, graphenes were poured or spin-coated on coupling agent-functionalized Ti for accelerating bone regeneration , yet it was inferior at in vivo osseointegration and drug delivery behavior. Titanate nanostructure with open pores on a titanium implant is favorable for cell growth and tissue . GO sheets were electrochemically deposited on titanium as mechanical hardener and surface activity regulator, demonstrating improved compressive strength and enhanced cell viability, proliferation, differentiation and osteogenic activities of human osteoblast-like cells . However, it is not easy to regulate the number of layers which play an important role in bio-functions and likely to come off from the substrate due to the weak bonding via physical interaction. In addition, in situ local delivery of drugs and growth factors within implants is desirable to achieve specific differentiation . For example, La et al. reported to load bone morphogenetic protein-2 (BMP-2), a well-known osteogenic factor, onto Ti substrates coated with GO, which enabled loading of large doses and the sustained release of the protein and promotes bone formation in vitro . However, BMP-2 may lose bioactivity over a short time due to its short half-life, which limits its local delivery, and does not always exhibit efficacy in bone defect repair in vivo . Dexamethasone (DEX) is a synthetic glucocorticoid usually applied in changing the expression levels of many proteins and enzymes contributed in bone differentiation . In this study, GO sheets were incorporated into a hydrothermally prepared porous titanate scaffold on Ti implants through a simple assembly method, which were used as DEX delivery platforms for enhancing osteogenic differentiation of rat bone marrow stem cells (rBMSCs) characteristics of self-renewal, differentiation potential, and pluripotency .
2
Experimental
2.1
Preparation of GO
GO was prepared by oxidation and exfoliation of commercially available graphite (Alfa Aesar) by modified Hummers method . Briefly, graphite flakes (Alfa Aesar) were pre-oxidized by concentrated H 2 SO 4 , K 2 S 2 O 8 , and P 2 O 5 by keeping the mixture at 80 °C for over 5 h. The mixture was then left to room temperature following by diluting with ultrapure water, filtering and washing thoroughly. The mixture was dried and re-oxidized by slowly adding H 2 SO 4 and KMnO 4 under 0 °C with stirring. The mixture was then kept at 35 °C for ∼2 h, and diluted slowly using ultrapure water. H 2 O 2 was added to the obtained solution till the color changed into brilliant yellow. The solution was then filtered and washed by diluted HCl and ultrapure water for several times. To remove the residue ions in the samples, the samples were dialyzed in ultrapure water for over 1 week. The resulted GO was dried at 60 °C in a vacuum oven overnight. The dried GO was exfoliated in water by ultrasonication for 1–2 h to obtain the single layer GO dispersion (1.5 mg/mL), characterized by atomic force microscopy (AFM).
2.2
Preparation and characterization of GO and rGO on Ti foils
Titanium foils (99.7% Ti TA2, BaoTi Group Co. Ltd., 7 × 7 × 0.8 mm 3 ) were mechanically polished by water sandpaper (grades 400, 600, 800 and 1000) sequentially and then ultrasonically cleaned with acetone, ethanol and ultrapure water successively. Nanonetwork-structured sodium titanate thin films were prepared on the surface of titanium foil by the alkali-hydrothermal reaction. A typical preparation process was as follows: Ti foils were immersed in 20 mL of 10 M NaOH aqueous solution at 60 °C for 24 h and washed repeatedly with ultrapure water . Then the titanium foils (remarked as Control and used as a reference material and a support for GO) were then immersed in 3% ethanol solution of 3-aminopropyltriethoxysilane (3-APTES, 3% ethanol solution of APTES) for 30 min, washed with ethanol and water sequentially, followed by blow-drying. GO was immobilized on Ti foil surface by immersing the functionalized samples into the GO solution (1.5 mg/mL) for 1 h, marked as GO-Ti.
RGO-Ti foils were obtained by immersing the GO-Ti foils into the 20% N , N -dimethylformamide (DMF, Sigma-Aldrich) solution of hydrazine (N 2 H 4 , Alfa Aesar) at 80 °C for 24 h , after which the color of the coverslips transformed from yellowish brown to grayish black due to the reduction of GO to rGO, marked as rGO-Ti.
GO-Ti and rGO-Ti samples were characterized by Raman spectroscopy (Jobin-Yvon HR 800, λ = 633 nm), X-ray photoelectron spectroscopy (XPS, ESCALAB 250), scanning electron microscope (SEM, Hitachi S-4800), Fourier transform infrared (FTIR, Perkin Elmer RXI), and a contact angle goniometer (JC2000C1).
2.3
DEX loading and release test in vitro
To obtain the dexamethasone loaded samples, Control, GO-Ti and rGO-Ti were immersed into 500 μL 1 mg/mL DEX solution at 37 °C for 24 h, respectively. The samples were then removed from the mixed solution, and gently washed with ultrapure water to remove non-adherent dexamethasone and dried in air. The samples were denoted as DEX-Control, DEX-GO-Ti and DEX-rGO-Ti, respectively. All the supernatant was collected and the amount was obtained using the DEX standard curve at 242 nm by ultraviolet–visible (UV–vis) spectroscopy (Hitachi U-3100) and the volume of supernatant. For calculation, it was subtracted from the total quantity, and then normalized by the area of the Ti foil.
In vitro drug release test was conducted using DEX-Control, DEX-GO-Ti and DEX-rGO-Ti immersed in PBS at 37 °C for 14 days. After incubation for 1, 3, 5, 7, 10, and 14 days, the released amount of DEX was obtained by the DEX standard curve at 242 nm using UV–vis spectroscopy and solution volume, and then normalized by the area of the Ti foil.
2.4
Cell culture
RBMSCs were isolated from femurs and tibias of 30-day-old neonatal male Wistar rats with a modified method originally described by Zhou et al. and cultured in medium containing 10% fetal bovine serum. RBMSCs were assessed by checking their surface markers using flow cytometry, demonstrating high purity according to CD45-negative (98.4%), CD54-(99.6%) were CD90-positive expression (94.1%) . After 3 passages (2 days for passage 1), rBMSCs were seeded on DEX-Control, DEX-GO-Ti, and DEX-rGO-Ti.
2.4.1
Cell proliferation
RBMSCs were on DEX-Control, DEX-GO-Ti and DEX-rGO-Ti for 1, 3, 5 days (n = 5 for each group). At each time point, the Cell Counting Kit-8 (CCK-8, Dojindo) was employed for quantitative evaluation of cell viability by monitoring absorbance of the formed formazan at 450 nm using a microplate reader (MULTISKAN MK3, Thermo). To intuitively observe cytoskeleton organization of rBMSCs for 3 days, immunofluorescence measurement of F-actin and nuclei was carried out through staining with phalloidin conjugated to Alexa Fluor 488 and Hoechst 33258 (Invitrogen) and examined under inverted fluorescence microscope (FV 300, Olympus).
2.4.2
Alkaline phosphatase (ALP) activity
After cultured on DEX-Control, DEX-GO-Ti and DEX-rGO-Ti for 7, 14 days. ALP activity of rBMSCs was measured using an ALP activity assay kit (Wako Pure Chemical Industries, Ltd.) and determined using the reaction of p -nitrophenol conversion to p -nitrophenylate at 37 °C for 15 min. The total amount of intracellular protein was detected by BCA protein assay kit (KeyGEN BioTECH) by measuring absorbance of the reaction solution at 570 nm. The relative ALP activity was normalized by the protein content of cells cultured on different samples.
2.4.3
Alizarin red S (ARS) staining
After 21 days, rBMSCs on DEX-Control, DEX-GO-Ti and DEX-rGO-Ti were fixed in 4% paraformaldehyde for 15 min, and then stained with 2% ARS (Sigma) at pH 4.2 for 10 min. After rinsing with distilled water three times, the samples were detected with a digital camera. Quantification of ARS staining was performed by elution with 10% (w/v) cetylpyridium chloride (Sigma) for 10 min and measurement of the absorbance at 570 nm (n = 3 for each group). The quantity was normalized to protein content on parallel samples.
2.4.4
Quantitative polymerase chain reaction (q-PCR) assay
After cultured on DEX-Control, DEX-GO-Ti and DEX-rGO-Ti for 7 and 14 days, the rBMSCs was treated with RNeasy Plus Mini Kit (Qiagen) to extract total RNA. The total RNA concentration and purity were determined using a spectrophotometer Q-5000 (Quwell) at 260/280 nm. Q-PCR analysis was performed using the LineGeneK (Hangzhou Bioer Technology Co., Ltd) for one housekeeping gene, glyceraldehyde-3-phosphate (GAPDH), and two targeting genes, osteopotin (OPN) and osteocalcin (OCN). Sequences of forward and reverse primers for tested genes were as description as follows: 5′-GCCTCGTCTCATAGACAAGATGGT-3′ and 5′-GAAGGCAGCCCTGGTAACC-3′ for GAPDH; 5′-TCCTGTCTCCCGGTGAAAGT-3′ and 5′-GGCTACAGCATCTGAGTGTTTGC-3′ for OPN; 5′-AAGCCCAGCGACTCTGAGTCT-3′ and 5′-CCGGAGTCTATTCACCACCTTACT-3′ for OCN. The relative transcript levels of the target gene expressions were normalized to GAPDH and expressed as mean ± S.D. (n = 3 for each group).
2.4.5
Immunofluorescence staining
After 14 days, rBMSCs were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min. They were permeablized using 0.1% Triton X-100 for 10 min and then blocked with 10% goat serum solution for 1 h at room temperature. After blocking, the cells were incubated overnight at 4 °C with the primary antibodies at 1:500 dilution against osteocalcin (mouse monoclonal anti-OCN, Abcam) and at 1:1000 dilution against osteopontin (rabbit polyclonal anti-OPN, Abcam). Goat anti-rabbit and goat anti-mouse secondary antibodies labeled by FITC at 1:200 dilutions in 5% goat serum solution was used for staining OPN and OCN for 1 h at room temperature, respectively. After rinsing the second antibody with PBS, cells were further labeled by Hoest 33258 for 5 min to stain the nuclei. Images of the stained samples were observed with an inverted fluorescence microscope.
2.5
Statistics analysis
The statistical analyses of all experimental data reported as means-standard deviations were performed using SPSS version 18.0 (SPSS, Inc.). Statistical comparisons were performed by one-way ANOVA, and p -values were considered statistically significant (*p < 0.05) and highly significant (**p < 0.01).
2
Experimental
2.1
Preparation of GO
GO was prepared by oxidation and exfoliation of commercially available graphite (Alfa Aesar) by modified Hummers method . Briefly, graphite flakes (Alfa Aesar) were pre-oxidized by concentrated H 2 SO 4 , K 2 S 2 O 8 , and P 2 O 5 by keeping the mixture at 80 °C for over 5 h. The mixture was then left to room temperature following by diluting with ultrapure water, filtering and washing thoroughly. The mixture was dried and re-oxidized by slowly adding H 2 SO 4 and KMnO 4 under 0 °C with stirring. The mixture was then kept at 35 °C for ∼2 h, and diluted slowly using ultrapure water. H 2 O 2 was added to the obtained solution till the color changed into brilliant yellow. The solution was then filtered and washed by diluted HCl and ultrapure water for several times. To remove the residue ions in the samples, the samples were dialyzed in ultrapure water for over 1 week. The resulted GO was dried at 60 °C in a vacuum oven overnight. The dried GO was exfoliated in water by ultrasonication for 1–2 h to obtain the single layer GO dispersion (1.5 mg/mL), characterized by atomic force microscopy (AFM).
2.2
Preparation and characterization of GO and rGO on Ti foils
Titanium foils (99.7% Ti TA2, BaoTi Group Co. Ltd., 7 × 7 × 0.8 mm 3 ) were mechanically polished by water sandpaper (grades 400, 600, 800 and 1000) sequentially and then ultrasonically cleaned with acetone, ethanol and ultrapure water successively. Nanonetwork-structured sodium titanate thin films were prepared on the surface of titanium foil by the alkali-hydrothermal reaction. A typical preparation process was as follows: Ti foils were immersed in 20 mL of 10 M NaOH aqueous solution at 60 °C for 24 h and washed repeatedly with ultrapure water . Then the titanium foils (remarked as Control and used as a reference material and a support for GO) were then immersed in 3% ethanol solution of 3-aminopropyltriethoxysilane (3-APTES, 3% ethanol solution of APTES) for 30 min, washed with ethanol and water sequentially, followed by blow-drying. GO was immobilized on Ti foil surface by immersing the functionalized samples into the GO solution (1.5 mg/mL) for 1 h, marked as GO-Ti.
RGO-Ti foils were obtained by immersing the GO-Ti foils into the 20% N , N -dimethylformamide (DMF, Sigma-Aldrich) solution of hydrazine (N 2 H 4 , Alfa Aesar) at 80 °C for 24 h , after which the color of the coverslips transformed from yellowish brown to grayish black due to the reduction of GO to rGO, marked as rGO-Ti.
GO-Ti and rGO-Ti samples were characterized by Raman spectroscopy (Jobin-Yvon HR 800, λ = 633 nm), X-ray photoelectron spectroscopy (XPS, ESCALAB 250), scanning electron microscope (SEM, Hitachi S-4800), Fourier transform infrared (FTIR, Perkin Elmer RXI), and a contact angle goniometer (JC2000C1).
2.3
DEX loading and release test in vitro
To obtain the dexamethasone loaded samples, Control, GO-Ti and rGO-Ti were immersed into 500 μL 1 mg/mL DEX solution at 37 °C for 24 h, respectively. The samples were then removed from the mixed solution, and gently washed with ultrapure water to remove non-adherent dexamethasone and dried in air. The samples were denoted as DEX-Control, DEX-GO-Ti and DEX-rGO-Ti, respectively. All the supernatant was collected and the amount was obtained using the DEX standard curve at 242 nm by ultraviolet–visible (UV–vis) spectroscopy (Hitachi U-3100) and the volume of supernatant. For calculation, it was subtracted from the total quantity, and then normalized by the area of the Ti foil.
In vitro drug release test was conducted using DEX-Control, DEX-GO-Ti and DEX-rGO-Ti immersed in PBS at 37 °C for 14 days. After incubation for 1, 3, 5, 7, 10, and 14 days, the released amount of DEX was obtained by the DEX standard curve at 242 nm using UV–vis spectroscopy and solution volume, and then normalized by the area of the Ti foil.
2.4
Cell culture
RBMSCs were isolated from femurs and tibias of 30-day-old neonatal male Wistar rats with a modified method originally described by Zhou et al. and cultured in medium containing 10% fetal bovine serum. RBMSCs were assessed by checking their surface markers using flow cytometry, demonstrating high purity according to CD45-negative (98.4%), CD54-(99.6%) were CD90-positive expression (94.1%) . After 3 passages (2 days for passage 1), rBMSCs were seeded on DEX-Control, DEX-GO-Ti, and DEX-rGO-Ti.
2.4.1
Cell proliferation
RBMSCs were on DEX-Control, DEX-GO-Ti and DEX-rGO-Ti for 1, 3, 5 days (n = 5 for each group). At each time point, the Cell Counting Kit-8 (CCK-8, Dojindo) was employed for quantitative evaluation of cell viability by monitoring absorbance of the formed formazan at 450 nm using a microplate reader (MULTISKAN MK3, Thermo). To intuitively observe cytoskeleton organization of rBMSCs for 3 days, immunofluorescence measurement of F-actin and nuclei was carried out through staining with phalloidin conjugated to Alexa Fluor 488 and Hoechst 33258 (Invitrogen) and examined under inverted fluorescence microscope (FV 300, Olympus).
2.4.2
Alkaline phosphatase (ALP) activity
After cultured on DEX-Control, DEX-GO-Ti and DEX-rGO-Ti for 7, 14 days. ALP activity of rBMSCs was measured using an ALP activity assay kit (Wako Pure Chemical Industries, Ltd.) and determined using the reaction of p -nitrophenol conversion to p -nitrophenylate at 37 °C for 15 min. The total amount of intracellular protein was detected by BCA protein assay kit (KeyGEN BioTECH) by measuring absorbance of the reaction solution at 570 nm. The relative ALP activity was normalized by the protein content of cells cultured on different samples.
2.4.3
Alizarin red S (ARS) staining
After 21 days, rBMSCs on DEX-Control, DEX-GO-Ti and DEX-rGO-Ti were fixed in 4% paraformaldehyde for 15 min, and then stained with 2% ARS (Sigma) at pH 4.2 for 10 min. After rinsing with distilled water three times, the samples were detected with a digital camera. Quantification of ARS staining was performed by elution with 10% (w/v) cetylpyridium chloride (Sigma) for 10 min and measurement of the absorbance at 570 nm (n = 3 for each group). The quantity was normalized to protein content on parallel samples.
2.4.4
Quantitative polymerase chain reaction (q-PCR) assay
After cultured on DEX-Control, DEX-GO-Ti and DEX-rGO-Ti for 7 and 14 days, the rBMSCs was treated with RNeasy Plus Mini Kit (Qiagen) to extract total RNA. The total RNA concentration and purity were determined using a spectrophotometer Q-5000 (Quwell) at 260/280 nm. Q-PCR analysis was performed using the LineGeneK (Hangzhou Bioer Technology Co., Ltd) for one housekeeping gene, glyceraldehyde-3-phosphate (GAPDH), and two targeting genes, osteopotin (OPN) and osteocalcin (OCN). Sequences of forward and reverse primers for tested genes were as description as follows: 5′-GCCTCGTCTCATAGACAAGATGGT-3′ and 5′-GAAGGCAGCCCTGGTAACC-3′ for GAPDH; 5′-TCCTGTCTCCCGGTGAAAGT-3′ and 5′-GGCTACAGCATCTGAGTGTTTGC-3′ for OPN; 5′-AAGCCCAGCGACTCTGAGTCT-3′ and 5′-CCGGAGTCTATTCACCACCTTACT-3′ for OCN. The relative transcript levels of the target gene expressions were normalized to GAPDH and expressed as mean ± S.D. (n = 3 for each group).
2.4.5
Immunofluorescence staining
After 14 days, rBMSCs were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min. They were permeablized using 0.1% Triton X-100 for 10 min and then blocked with 10% goat serum solution for 1 h at room temperature. After blocking, the cells were incubated overnight at 4 °C with the primary antibodies at 1:500 dilution against osteocalcin (mouse monoclonal anti-OCN, Abcam) and at 1:1000 dilution against osteopontin (rabbit polyclonal anti-OPN, Abcam). Goat anti-rabbit and goat anti-mouse secondary antibodies labeled by FITC at 1:200 dilutions in 5% goat serum solution was used for staining OPN and OCN for 1 h at room temperature, respectively. After rinsing the second antibody with PBS, cells were further labeled by Hoest 33258 for 5 min to stain the nuclei. Images of the stained samples were observed with an inverted fluorescence microscope.
2.5
Statistics analysis
The statistical analyses of all experimental data reported as means-standard deviations were performed using SPSS version 18.0 (SPSS, Inc.). Statistical comparisons were performed by one-way ANOVA, and p -values were considered statistically significant (*p < 0.05) and highly significant (**p < 0.01).
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