Gelatin-Derived GrapheneâSilicate Hybrid Materials Are

Research Article www.acsami.org

Gelatin-Derived Graphene−Silicate Hybrid Materials Are Biocompatible and Synergistically Promote BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells Yulong Zou,†,‡,†† Nader Taheri Qazvini,§,∥,†† Kylie Zane,§ Monirosadat Sadati,§,∥ Qiang Wei,‡,⊥ Junyi Liao,‡,⊥ Jiaming Fan,‡,⊥ Dongzhe Song,‡,# Jianxiang Liu,‡,¶ Chao Ma,‡,∇ Xiangyang Qu,‡,⊥ Liqun Chen,‡,⊥ Xinyi Yu,‡,⊥ Zhicai Zhang,‡,¶ Chen Zhao,‡,⊥ Zongyue Zeng,‡,⊥ Ruyi Zhang,‡,⊥ Shujuan Yan,‡,⊥ Tingting Wu,‡,∇ Xingye Wu,‡,⊥ Yi Shu,‡,⊥ Yasha Li,‡,⊥ Wenwen Zhang,‡,○ Russell R. Reid,‡,◆ Michael J. Lee,‡ Jennifer Moritis Wolf,‡ Matthew Tirrell,§,∥ Tong-Chuan He,‡,⊥ Juan J. de Pablo,*,§,∥ and Zhong-Liang Deng*,† †

Department of Orthopaedic Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States § Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States ∥ Argonne National Laboratory, Argonne, Illinois 60439, United States ⊥ Ministry of Education Key Laboratory of Diagnostic Medicine, The Affiliated Hospitals of Chongqing Medical University, Chongqing 400016, China # Department of Conservative Dentistry and Endodontics, West China School of Stomatology, Sichuan University, Chengdu 610041, China ¶ Department of Orthopaedic Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430022, China ∇ Departments of Neurosurgery and Otolaryngology-Head & Neck Surgery, The Affiliated Zhongnan Hospital of Wuhan University, Wuhan 430071, China ○ Department of Laboratory Medicine and Clinical Diagnostics, the Affiliated Yantai Hospital, Binzhou Medical University, Yantai 264100, China ◆ Department of Surgery, Section of Plastic Surgery, The University of Chicago Medical Center, Chicago, Illinois 60637, United States ‡

S Supporting Information *

ABSTRACT: Graphene-based materials are used in many fields but have found only limited applications in biomedicine, including bone tissue engineering. Here, we demonstrate that novel hybrid materials consisting of gelatin-derived graphene and silicate nanosheets of Laponite (GL) are biocompatible and promote osteogenic differentiation of mesenchymal stem cells (MSCs). Homogeneous cell attachment, long-term proliferation, and osteogenic differentiation of MSCs on a GL-scaffold were confirmed using optical microscopy and scanning electron microscopy. GL-powders made by pulverizing the GL-scaffold were shown to promote bone morphogenetic protein (BMP9)-induced osteogenic differentiation. GL-powders increased the alkaline phosphatase (ALP) activity in immortalized mouse embryonic fibroblasts but decreased the ALP activity in more-differentiated immortalized mouse adipose-derived cells. Note, however, that GL-powders promoted BMP9-induced calcium mineral deposits in both MSC lines, as assessed using qualitative and quantitative alizarin red assays. Furthermore, the expression of chondro-osteogenic regulator markers such as Runx2, Sox9, osteopontin, and osteocalcin was upregulated by the GL-powder, independent of BMP9 continued...

Received: January 6, 2017 Accepted: April 13, 2017 Published: April 13, 2017 © 2017 American Chemical Society

15922

DOI: 10.1021/acsami.7b00272 ACS Appl. Mater. Interfaces 2017, 9, 15922−15932

Research Article

ACS Applied Materials & Interfaces

stimulation; although the powder synergistically upregulated the BMP9-induced Osterix expression, the adipogenic marker PPARγ was unaffected. Furthermore, in vivo stem cell implantation experiments demonstrated that GL-powder could significantly enhance the BMP9-induced ectopic bone formation from MSCs. Collectively, our results strongly suggest that the GL hybrid materials promote BMP9-induced osteogenic differentiation of MSCs and hold promise for the development of bone tissue engineering platforms. KEYWORDS:

graphene, Laponite, bone morphogenetic protein, mesenchymal stem cells, bone tissue engineering,

1. INTRODUCTION Successful bone tissue engineering requires at least three key elements: osteoprogenitors, growth factors, and scaffolds.1,2 An ideal scaffold material should be both osteoinductive and osteoconductive. Most of the currently used scaffold materials include bioactive glasses, calcium phosphates such as hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP), and biopolymers,3−7 many of which only act as osteoconductive scaffolds for the in-growth of progenitors but lack osteoinductivity to drive osteoprogenitor differentiation.1,6 Therefore, there is an urgent need to develop novel scaffold materials that can effectively couple both osteoinductive and osteoconductive processes for bone tissue engineering. Graphene is comprised of a single layer of carbon atoms. Since its original synthesis, it has been used extensively in chemistry, physics, and materials science. Applications in biomedicine, particularly regenerative medicine, have been limited.8−10 It has been reported that a single-layer graphene sheet can direct human mesenchymal stem cells (hMSCs) to undergo osteogenic differentiation and that this effect is comparable to that of bone morphogenetic protein-2 (BMP2).11 Moreover, synthetic silicates are novel nanomaterials that can dissociate into Si(OH)4 and lithium, sodium, and magnesium cations under physiological conditions and have a significant impact on the cell behavior. Among synthetic silicates, Laponite is biocompatible and is able to induce osteogenic differentiation of stem cells, even in the absence of other factors.12,13 Several types of mesenchymal stem cells (MSCs), which are common sources of osteoblastic progenitors, have been isolated from different sources.14−17 Although various growth factors have been used to promote osteogenesis, the members of the transforming growth factor (TGF)-β superfamily of BMPs are the only group of biofactors that can induce de novo bone formation from MSCs and, as such, hold the greatest potential for bone tissue engineering.18−20 Although BMP2 and BMP7 were approved by the Food and Drug Administration (FDA) to treat spine fusion and fracture nonunion, we previously demonstrated that, among the 14 types available, the least studied protein, namely BMP9, turned out to be the most potent osteogenic BMP.19−25 Thus, more attention should be paid to the application of BMP9 in bone tissue engineering. To assess the optimal conditions for bone formation and bone tissue engineering, we have established a panel of reversibly immortalized MSC lines, including immortalized mouse embryonic fibroblasts (iMEFs), immortalized mouse adipose-derived cells (iMADs), and immortalized mouse calvarial cells (iCALs), which have been shown to successfully undergo osteogenic differentiation and form robust trabecular bone in vivo when stimulated with BMP9.26−30 Our studies indicate that these MSC lines represent different stages of multipotency of MSCs, with iMEFs being at the top of the hierarchy, followed by iMADs and then iCALs.26−30 In this study, we propose a new class of graphene/Laponite (GL) hybrid materials, which we fabricated in two forms: a porous scaffold (GL-scaffold) and a fine powder (GL-powder).

We have investigated their potential biomedical applications for bone healing. To assemble Laponite nanosheets and graphenelike species on the nanometric scale, we use a simple, environmentally friendly, and cost-effective synthesis procedure consisting of Laponite/gelatin complexation,3 followed by molding, lyophilization, and thermal treatment in the absence of oxygen at a high temperature. We analyzed the effect of the hybrids on the proliferation and differentiation of MSCs and found that the GL-scaffold is biocompatible, yields homogeneous attachment and supports long-term proliferation of MSCs. Furthermore, the GL-scaffold was shown to promote apparent osteogenic differentiation of MSCs in the absence of other osteogenic inducers. The GL-powder was found to effectively promote BMP9-induced osteogenic differentiation of MSCs. Stem cell implantation experiments further demonstrate that the GL-powder is able to significantly enhance BMP9-induced ectopic bone formation from MSCs, indicating that the inclusion of the GL-powder with BMP9-transduced MSCs can lead to a more robust bone formation in vivo, a feature that was mechanistically supported by our in vitro studies. Thus, our results demonstrate that the GL hybrids proposed here hold promise for applications in bone tissue engineering.

2. MATERIALS AND METHODS 2.1. Synthesis and Structural Characterization of GL Hybrids. Gelatin (type A, porcine skin, 300 bloom) was purchased from SigmaAldrich, USA. Silicate nanosheets (Laponite XLG, 1 nm thick and ∼30 nm in diameter) were obtained as gift sample from BYK Additives Inc., Gonzales, Texas. Milli-Q water (Millipore, MA, USA) was used for all experiments. All materials were used without further purification. The GL hybrids were prepared by the complexation of aqueous gelatin and Laponite dispersions, followed by carbonization. A typical synthesis is as follows. A stock solution of 2% (w/v) gelatin in water was prepared at 40 °C. An optically transparent dispersion of Laponite nanosheets [1.5% (w/v), pH = 9.8] was prepared by slowly adding Laponite to water at room temperature and stirring at 1000 rpm for 2 h, followed by ultrasonication using an ultrasonic homogenizer (Branson Sonifier S-450A) for 3 min. Gelatin/Laponite complexes were prepared by mixing designated amounts of Laponite dispersion, gelatin solution, and water at 250 rpm for 45 s. The weight ratio of Laponite:gelatin was fixed at 1:1; 1:2; 1:5; or 1:10. The mixtures were cast in polyethylene dishes and stored at 4 °C overnight and then transferred to the freezer at −80 °C. Gelatin/Laponite aerogels were prepared by freeze-drying at −4 °C for 48 h. The aerogels were then transferred to a tube furnace and heated at a rate of 10 °C min−1 to 800 °C under an ultrahigh pure nitrogen atmosphere and carbonized for 2 h at this temperature to obtain gelatin-derived carbon/Laponite hybrid aerogels (GL-scaffolds). GL-powders were prepared by grinding the GL-scaffolds. The samples were denoted as C[GEL/L = X], where X represents the ratio of gelatin (GEL) to Laponite, and C indicates carbonized. The GL hybrid materials were characterized using transmission electron microscopy (TEM, FEI Tecnai F30, accelerating voltage of 200 kV), scanning electron microscopy (SEM, Nova NanoSEM 230, USA), Raman spectroscopy (Horiba LabRAM HR Evolution) with the laser excitation at 633 nm, X-ray photoelectron spectroscopy (XPS, Kratos AXIS Nova), and an Instron 5800 Materials Tester at a strain rate of 5%/min up to 40% strain. The noncarbonized gelatin/ Laponite samples were also analyzed using a thermogravimetric analyzer 15923


Research Article


2.8. SEM Detection of In Vitro Biomineralization. To determine the ability of GL hybrids to assist biomineralization, the samples were immersed in 10× simulated body fluid (SBF) solution for 6 h and then air-dried for surface characterization using SEM and Raman spectroscopy. 2.9. Touchdown qPCR (TqPCR) Analysis. Subconfluent iMADs were seeded in 60 mm dishes and infected with Ad-GFP or Ad-BMP9 in the presence or absence of GL-powder. Forty-eight hours after the infection, the cells were treated with TRIzol Reagents (Invitrogen). The extracted total RNA was purified with chloroform and precipitated using isopropanol, followed by reverse transcription using random hexamer primers and M-MuLV Reverse Transcriptase (New England Biolabs, Ipswich, MA). After 10- to 50-fold dilution, the consequent products were used as cDNA templates for PCR reaction. Primer3Plus was used to design TqPCR primers (usually 18−20 mers, product sizes in the range of 120−200 bp) (Table S1).27 TqPCR analysis was carried out as described.28,29 The brief TqPCR reaction protocol is as follows: 95 °C 3 min ×1; 95 °C 20 s, 66 °C 10 s ×4, by decreasing 3 °C/cycle; 95 °C 20 s, 55 °C 10 s, 70 °C 1 s, and recorded through SYBR Green I channel for 40 cycles. The expression of all genes was normalized to Gapdh. Each TqPCR reaction was performed in triplicate. 2.10. Ectopic Bone Formation, Microcomputed Tomographic (μCT) Imaging, and Histological Analysis. The use and care of animals in this study were approved by the Institutional Animal Care and Use Committee (IACUC). The animal-related experimental protocols were performed according to the guidelines of IACUC. The subcutaneous stem cell implantation was performed as described.4,24,30,31 Briefly, the iMEF cells were infected with Ad-BMP9 or Ad-GFP. Twentyfour hours after the infection, the cells were collected and mixed with or without GL-powder in suspension. The mixture was subcutaneously injected into athymic nude mice (n = 5/group, 5−6 week old, female; Envigo/Harlan Research Laboratories; 2 × 106 cells per site). Four weeks post implantation, all mice were euthanized, and the ectopic masses were retrieved from the injection sites, fixed in 10% formalin, and imaged using the micro-CT system (GE Healthcare, Preclinical trimodality). Amira 5.3 from Visage Imaging Inc. was used to analyze the image data. Reconstructed three-dimensional (3D) volumetric quantitative data were determined as previously described.22,26,32,33 After μCT imaging, the retrieved specimens were decalcified for 3 days and subjected to paraffin-embedding. Continuous 5 μm sections of these paraffinized masses were deparaffinized and then subjected to histological staining as described.13,34,35 Trabecular bone area measurement was recorded using the ImageJ program. 2.11. Statistical Analysis. ALP activity and TqPCR assays were performed in three independent batches and/or conducted in triplicate. Statistical analysis was carried out using the Microsoft Excel program. Data were expressed as mean ± SD. One-way analysis of variance (ANOVA) and student t test were used to determine the statistical significance with a cutoff of p < 0.05.

(TA Instruments, Q600 SDT Simultaneous DSC-TGA) at a heating rate of 10 °C min−1 in a 50 mL/min nitrogen flow. 2.2. Cell Culture and Related Chemicals. The MSC lines iMADs, iMEFs, and iCALs were previously characterized.4−7 These MSC lines and HEK-293 (ATCC, Manassas, VA) and 293pTP cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 100 U mL−1 penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Thermo Fisher Scientific (Waltham, MA), unless indicated otherwise. 2.3. Preparation of Recombinant Adenoviruses. AdEasy technology was used to generate recombinant adenoviruses as described.8−10 The coding region of human BMP9 was polymerase chain reaction (PCR)-amplified and cloned into an adenoviral shuttle vector and subsequently used to generate recombinant adenoviruses in HEK-293 or 293pTP cells.11,12 The resultant adenoviruses were designated as Ad-BMP9, which also express green fluorescent protein (GFP) as the marker for monitoring the infection efficiency.13,14 An analogous adenovirus expressing only GFP (Ad-GFP) was used as the vector control.15−17 Polybrene with a concentration of 4−8 μg/mL was used to promote infection efficacy in all experiments using adenoviruses.18 2.4.1. MSC Culturing on GL-Scaffold. GL-scaffolds were prepared as 8 mm3/cube, disinfected with 70% ethanol, incubated in 0.1% gelatin for 1 h, and dried before culturing, as described.19 MSCs were infected using adenovirus vectors expressing GFP (Ad-GFP). At 24 h post infection, the cells were trypsinized and resuspended in complete DMEM at a concentration of 2 × 104/μL. A total of 50 μL of volume cell suspension was seeded onto each GL-scaffold and incubated for 2 h in an incubator, and then, the GL-scaffolds were transferred into 24-well plates and cultured in complete DMEM. 2.4.2. Culturing MSCs with GL-Powder. The MSCs were infected using Ad-BMP9 or Ad-GFP and cultured with GL-powder at a ratio of 1 mg: 2 × 106 cells in complete DMEM. 2.4.3. Gaussia Luciferase (GLuc) Activity Assay. AdR-GLuc was used to infect subconfluent iMADs. Twenty-four hours after infection, iMAD cells were harvested, resuspended, seeded onto the GL-scaffold, and maintained in complete DMEM in a 24-well plate. On days 1, 2, 3, 5, and 7 after culture, a volume of 50 μL was taken from the medium for the GLuc activity assay. BioLux Gaussia Luciferase Assay Kit (New England Biolabs) was used as described.20,21 Each assay condition was carried out in triplicate. 2.5. SEM Analysis. The MSCs/GL-scaffolds underwent primary fixation in 2.5% glutaraldehyde buffered with 0.1 M sodiumcacodylate (NaC) solution overnight at 4 °C. The samples were then rinsed with 0.1 M NaC, underwent secondary fixation with 1% osmium tetroxide in 0.1 M NaC for 1 h, dehydrated with gradual ethanol washes for 15 min each (30, 50, 75, 85, 95, and 100% three times), dried in a critical point dryer (Leica EM CPD300), and finally mounted onto an aluminum SEM pin stub mount using a double-sided carbon tape. The samples were imaged using SEM (Nova NanoSEM 230, USA) with an accelerating voltage of 5 kV. 2.6. ALP Activity Assay. A modified assay using the Great EscAPe SEAP Chemiluminescence assay kit (BD Clontech) was used to assess the ALP activity quantitatively.22−24 Each ALP assay was carried out in triplicate. 2.7. Alizarin Red S Staining and Quantification Assay for Matrix Mineralization. Subconfluent MSCs were infected with AdGFP or Ad-BMP9 and cultured in 24-well plates in complete DMEM containing β-glycerophosphate (10 mM) and ascorbic acid (50 μg/mL). Ten days post infection, alizarin red S was used to stain the calcium deposit of the matrix mineralization, as described.25,26 In brief, iMADs and iMEFs were fixed with glutaraldehyde (0.05%) at 25 °C for 10 min, washed with phosphate-buffered saline (PBS, pH = 4.2), followed by incubation with 0.4% alizarin red S at 37 °C for 5 min, and being extensively washed with PBS (pH = 4.2). The red stains of the mineralized nodules of the cell matrix were observed under a bright field microscope. Each assay condition was carried out in triplicate. Alizarin red quantification was carried out using the ImageJ program.

3. RESULTS 3.1. Structural Characterization of GL Hybrids. The carbonization process and the composition of the obtained GL hybrids were assessed using thermogravimetric analysis (TGA) under N2 flux. The carbonization appeared to be complete below 800 °C (Figure 1a), confirming that the adopted experimental conditions were adequate for the preparation of GL hybrids. In the TGA results, the 6−8% weight loss up to 200 °C is attributed to dehydration, and the weight losses at 300−750 °C are due to degradation of gelatin. No transformation of Laponite was observed below 800 °C. Compared with the decomposition profile of pure gelatin, one can conclude that complexation with Laponite nanosheets increases the thermal stability of the polymer. Quantitatively, almost 25% of the polymer is converted into carbonaceous material and incorporated into the GL hybrids. Therefore, the C[GEL/L = 5] sample, for instance, consists of 57 wt % carbonaceous material and 43 wt % Laponite nanosheets (Figure 1a). Under uniaxial compression, the GL 15924


Research Article


We further conducted XPS to determine the elements and their chemical states in the GL hybrids (Figure 1f). The survey spectrum of C[GEL/L = 2] shows the presence of O (approximately 531 eV), Si (approximately 100 and 150 eV), C (approximately 285 eV), and other elements present in the Laponite structure including Li, Ca, Mg, and Na (Figure 1f). The C1s spectrum is deconvoluted into four components (Figure 1g). The strong peak at 284.8 eV is ascribed to the graphitic carbon (sp2 hybridization), whereas the weak peaks at higher binding energies may be attributed to the carbon combined with other elements with high electronegativity, such as N and O. Specifically, the peak at 285.6 eV corresponds to the carbons remaining from the gelatin C−N bonds. The peaks at 286.9 and 288.9 eV can be assigned to the carbon on C−O bonds and CO bonds, respectively. Quantitatively, approximately 66% of the carbonaceous material is composed of C−C bonds, which implies the relatively low defect of carbon in the GL hybrids and the formation of graphene-like structures (Figure 1g). The formation of graphene was confirmed using Raman spectroscopy. The Raman spectrum of the GL hybrids revealed the characteristic G-band (graphitic band) between 1580 and 1600 cm−1, ascribed to the E2g vibrational mode and a D-band (defect band) at 1330−1340 cm−1, associated with the defectactivated breathing modes of the A1g symmetry of the aromatic rings (Figure 1h). These bands along with the 2D-band (2500− 2900 cm−1) are well-known to be indicative of graphene. The intensity ratio ID/IG of the D and G bands provides information on the disordered structure.37 The intensity ratio increased with increasing Laponite content in the GL hybrids and changed from 0.95 for pure gelatin carbonized in the absence of Laponite, C[GEL], to 1.07 for the C[GEL/L = 1] sample. The higher ID/IG ratios indicate increases in the structural defects and bonding disorder probably because of the existence of a significant amount of pores and edges in the carbonaceous materials formed in the presence of Laponite nanosheets. 3.2. In Vitro Biomineralization. To evaluate the bonebonding ability of the GL-scaffolds, we analyzed the in vitro biomineralization by submerging the scaffolds in SBF and deposition of apatite-like deposits on their surfaces. SEM and Raman spectroscopy clearly showed the formation of the hydroxyapatite (HAp) layer on the surface of C[GEL/L = 2] after 6 h of immersion in 10× SBF (Figure 1i,j). The evolution of vibrational bands at 430−450 and 970 cm−1 corresponding to ν2 PO43− and ν1 PO43− domains of HAp38 are shown in Figure 1i. On the basis of these results, the GL hybrids can be considered as promising bioactive materials for bone tissue regeneration. 3.3. Cell Attachment, Morphology, and Proliferation on the GL-Scaffold. We further tested the biocompatibility of the GL-scaffold (C[GEL/L = 2]). Three MSC lines, iMADs, iMEFs, and iCALs, were infected with Ad-GFP, seeded on the surface of the scaffold and maintained in complete DMEM (Figure 2B(c)). On day 1, a homogeneous attachment of cells on the scaffold was observed in all three lines. We noticed that some cells were distinct in shape, whereas some were not, indicating that the cells may attach to both the outer and inner faces of the scaffold. On day 7, all three lines were found viable, and cell proliferation was clearly observed (Figure 2A). We then continued culturing and found that the GFP signal dropped quickly as the cells proliferated and divided (Figure 2B(a)). On day 13, when another dose of Ad-GFP was added to the medium, the cells were able to be reinfected as the GFP signal was reintensified on day 15 (Figure 2B(b)), indicating that the infected cells on the scaffold were still viable and maintained a

Figure 1. Structural characterization of gelatin-derived GL hybrids. (a) Thermogravimetric analyzer curves of gelatin/Laponite, [GEL/L], hybrids under N2 flux. (b) Typical morphology of [GEL/L] hybrids. (c) SEM image of the C[GEL/L = 2] scaffold. (d) TEM image of the C[GEL/L = 2] powder. Red arrows show the Laponite nanosheets. (e) Mesoporous structure of graphene-like layers in the C[GEL/L = 2] powder. (The inset shows a photograph of the C[GEL/L = 2] scaffold standing on a spider plant.) (f) XPS survey spectrum of the C[GEL/L = 2] scaffold. (g) XPS C1s high-resolution spectrum of C[GEL/L = 2]. (h) Raman spectra of GL-scaffolds with different compositions. (i) Raman spectra showing the evolution of HAp characteristic peaks on the C[GEL/L = 2] scaffold immersed in 10× SBF. (j) SEM images showing the deposition of minerals on a GL-scaffold after 6 h of immersion in 10× SBF. Representative images are shown.

hybrid scaffolds sustain large-strain deformations (ε > 40%) and show a compressive modulus of 8−10 MPa. SEM images reveal the porous structure of the freeze-dried gelatin/Laponite hybrids before carbonization (Figure 1b) and the preservation of the 3D porous structure following carbonization (Figure 1c). The size and distribution of pores within the porous scaffolds play an important role in their ability to host cells and direct their distribution throughout the structure.36 The GL hybrids displayed pore sizes between 50 and 100 μm, which remained nearly unchanged after carbonization. Furthermore, TEM images verified that the Laponite nanosheets are uniformly dispersed within the carbonaceous structure and form no microscopically visible aggregates (Figure 1d). The TEM images also confirmed the twodimensional (2D) sheetlike structure of the carbonaceous material in the form of nanoparticles and the presence of mesopores in their structure (Figure 1e). 15925


Research Article


Figure 2. Attachment, proliferation, and morphology of MSCs on the GL-scaffolds. (A) iMADs, iMEFs, and iCALs exhibited a homogeneous attachment on day 1 and remained viable at day 7. (B(a,b)) iMEFs maintained proliferative after 15 days. On day 13, a second dose of Ad-GFP was added to the medium, and the GFP signal was observed on day 15. (B(c)) MSCs were infected with Ad-GFP and seeded on the surface of the scaffold and maintained in complete DMEM. (C) iMADs were infected with AdR-GLuc and seeded onto the GL-scaffolds; at the indicated time points, the culture medium was taken for GLuc assay. Each assay was carried out in triplicate. Each experimental condition was set up in triplicate.

that all three lines cultured on the scaffold showed numerous well-mineralized nodules with many mineral particles observed on the surface of the scaffold (Figure 3B). 3.5. Synergistic Augmentation of BMP9-Induced Osteogenic Differentiation of Stem Cells by GL-Powder. Then, we tested the osteoinductivity of the GL-powder in iMEFs and iMADs in the presence or absence of BMP9 stimulation. We found that in both MSC lines, GL-powder alone induced negligible ALP activity, whereas BMP9 alone induced robust ALP activities (Figure 4A). When iMADs were infected with AdBMP9, the ALP activity was shown to decrease in the presence of GL-powder (Figure 4A(a)). On the contrary, when cultured with Ad-BMP9, the ALP activity of iMEFs was increased by the GL-powder on days 3, 5, and 7 (Figure 4A(b)). This opposite effect of GL-powder on iMADs vs iMEFs may be caused by the fact that iMADs are more differentiated cells compared with iMEFs, so that the GL-powder treatment may further accelerate the BMP9-induced osteogenic differentiation process, leading to a decrease in the early osteogenic marker ALP activity. However, the exact mechanism(s) remains to be fully understood. Nonetheless, alizarin red S staining and quantification showed that the GL-powder significantly promoted matrix mineralization in both iMADs and iMEFs when treated with Ad-BMP9 but failed to do so when Ad-BMP9 was absent (Figure 4B,C). Hence, these results indicate that the GL-powder may accelerate BMP9-induced terminal osteogenic differentiation of MSCs.

high proliferative capability after more than 2 weeks of culture because adenoviruses primarily infect actively dividing cells. To monitor cell proliferation quantitatively, we took advantage of a GLuc expressing adenovirus, AdR-GLuc.39,40 The iMAD cells were transduced with AdR-GLuc and cultured on GL-scaffolds (Figure 2C(a)). On days 1, 2, 3, 5, and 7 post transduction, GLuc activity was quantitatively assessed, and we found that iMAD cells exhibited a stable level of GLuc activity during the first 3 days, increased on day 5, and slightly decreased on day 7, probably as a result of the degradation of adenoviruses (Figure 2C(b)). The result further confirms that the cells on the GL-scaffold were viable and proliferative. The cell morphology and interactions with the scaffold were confirmed using both microscopy techniques. The iMADs and iMEFs had an elongated morphology, whereas iCALs adapted a relatively round morphology. More morphological details were further revealed using SEM because the MSCs populated on both the outer and the inner faces of the scaffold, indicating that the GL-scaffold is highly osteoconductive. On the other hand, the iMADs had many long protrusions stretched out from the cell body that crossed the micropores of the scaffold and attached to the nearby surface (Figure 3A). 3.4. Induction of Osteogenic Differentiation of the MSCs and Enhancement of Matrix Mineralization by the GL-Scaffold. MSC-seeded scaffolds were cultured in a mineralization medium, and SEM was performed on day 15. We found 15926


Research Article


Figure 3. Effect of GL-scaffolds on the cell morphology and mineralization of MSCs observed using SEM. (A) iMADs, iMEFs, and iCALs’ morphology and interaction with the GL-scaffolds. Cells were fixed on day 7 for SEM imaging. MSCs are indicated by arrows. Representative images are shown. (B) MSCs seeded on the scaffolds and cultured for 15 days in the mineralization medium before SEM imaging. The well-mineralized matrix with many mineral nodules on the surface of the cells and scaffolds are indicated by arrows. Representative images are shown.

Because MSCs are able to differentiate into different lineages, we used the iMADs to test the expression of the markers for osteogenic, chondrogenic, and adipogenic lineages when cultured with the GL-powder. The expression of chondrogenic and osteogenic markers, Sox9 and Runx2, but not adipogenic marker PPARγ, was significantly increased by the GL-powder (Figure 5A). Consistent with our previous observation,41−43 the induction of the Runx2 expression was not apparent at 48 h after Ad-BMP9 infection, although Ad-BMP9 induced a robust PPARγ expression. Furthermore, the expression of Runx2 downstream target gene Osterix was induced by the GL-powder, which was potentiated in the presence of BMP9 (Figure 5B(a)). Later bone markers osteopotin (Opn) and osteocalcin (Ocn) were also significantly upregulated by the GL-powder independent of BMP9 stimulation (Figure 5B(b,c)). Taken together, these results demonstrate that the GL-powder can induce osteogenic differentiation to a certain extent by itself and/or act synergistically to promote BMP9-induced osteogenic differentiation of MSCs. 3.6. Enhancement of BMP9-Induced Ectopic Bone Formation and Mineralization of Bony Masses by GL-Powder. To further verify our in vitro results, we continued to test the effect of GL-powder on BMP9-induced ectopic ossification in an ectopic bone formation animal model. When iMEFs

were infected using Ad-BMP9 or Ad-GFP and mixed without or with GL-powder (N = 5 for each group), we found that the overall sizes did not differ significantly among the bony masses recovered from iMEFs + BMP9 and iMEFs + BMP9 + GL-powder (Figure 6A(a)), whereas no detectable masses were retrieved from the Ad-GFP-transduced iMEFs only group and the iMEFs + GL-powder group (Figure 6A(b)). Consistent with our previous reports, histological evaluation revealed that BMP9 induced a robust bone formation of iMEFs, although numerous yet-to-be differentiated MSCs were readily detectable (Figure 6B(a)). However, in the group of the BMP9-transduced iMEFs mixed GL-powder, we found more trabecular bone structure (Figure 6B(b)), which was also confirmed by the quantitative measurement of the trabecular bone area (Figure 6B(c)), indicating that the inclusion of GL-powder with BMP9transduced MSCs can lead to a more robust bone formation in vivo, which was mechanistically supported by our in vitro studies.

4. DISCUSSION There have been unmet clinical demands for effective bone tissue engineering to repair large bone defects and nonunion fractures. In the past decades, significant progress has been made in identifying MSC sources and/or growth factors for bone regeneration. Although significant efforts have been devoted to 15927


Research Article


Figure 4. Effect of GL-powder on the ALP activity and matrix mineralization of MSCs. (A) GL-powder decreases BMP9-induced ALP activity of iMADs (a), whereas enhances that of iMEFs (b). The Cells were infected with Ad-GFP or Ad-BMP9, and the relative ALP activity was quantitatively determined on days 3, 5, and 7 after infection. Assays were done in triplicate. (B,C) GL-powder promotes BMP9-induced matrix mineralization of both iMADs (a) and iMEFs (b). The cells were infected with Ad-GFP or Ad-BMP9, cultured in a mineralization medium for 10 days, and stained with alizarin red S. Alizarin red S quantification was done using the ImageJ program. Assays were carried out in triplicate. Representative images are shown. “**”, p < 0.05 when compared with the BMP9 group; “*”, p < 0.05 when compared with the GFP group.

the development of various scaffolds, ideal scaffolds that possess both osteoinductive and osteoconductive capabilities and thus induce effective bone in-growth at the repair sites are still lacking. In this study, we fabricated novel GL hybrid materials in porous scaffold (GL-scaffold) and fine powder (GL-powder) forms. To assemble Laponite nanosheets and graphene-like species on the nanometric scale, we used a simple, environmentally friendly, and cost-effective synthesis procedure consisting of Laponite/gelatin complexation,3 followed by molding, lyophilization, and thermal treatment in the absence of oxygen at 800 °C. The gelatin-derived graphene-like materials have been used in many areas including advanced sensors, CO2 adsorbents, and electrode materials in lithium ion batteries, supercapacitors, and oxygen reduction reaction in fuel cells.44−48 However, to the best of our knowledge, this is the first report on the use of Laponite nanosheets to obtain GL hybrids based on gelatin for biomedical applications. By testing the hybrids’ impact on the proliferation and differentiation of stem cells, we found that the GL-scaffold is biocompatible and can provide homogeneous attachment, provide long-term proliferation, and promote spontaneous osteogenic differentiation of MSCs without any osteogenic inducers, whereas the GL-powder can promote the BMP9-induced osteogenic differentiation of MSCs.

Thus, these novel GL hybrids hold great potential for bone engineering. Three-dimensional graphene materials have many applications, whereas their potentials in bone tissue engineering have not been well-studied yet. Consistent with our study, Crowder et al. found that their 3D graphene foam could promote osteogenic differentiation of human MSCs (hMSCs).49 Also, iMADs and iMEFs on our GL-scaffold have an elongated morphology similar to that of hMSCs on graphene foam and are also identical to that of hMSCs undergoing osteogenic differentiation on TiNTs.50 This morphological change further confirmed that the stem cells undergo osteogenic differentiation on the GL-scaffold. One of the underlying mechanisms through which the GL-scaffold promotes osteogenic differentiation may be at least in part attributable to the materials’ surface topography. A graphene-incorporated CS (chitosan substrata) substrate was previously fabricated, and it was found that nanoscale topographical cue and its stiffness and roughness had a significant impact on adhesion and differentiation of stem cells.51 Akhavan et al. also reported that their graphene nanogrids could accelerate osteogenic differentiation of stem cells, and this effect is caused by physical stresses induced on the cells by the surface topographic features of the nanogrids.52 These experiments, along 15928


Research Article


Figure 5. Effect of GL-powder on the expression of osteogenic markers. (A) GL-powder upregulates the expression of Sox9 and Runx-2, whereas it has a limited impact on the PPARγ expression. Subconfluent iMADs cells were infected with Ad-GFP or Ad-BMP9, cultured with or without GL-powder. Total RNA was isolated at 48 h and subjected to TqPCR analysis using gene-specific primers for mouse Runx2, PPARγ, and Sox9. (B) GL-powder promotes the expression of osteogenic markers Osx, Ocn, and Opn. Subconfluent iMAD cells were infected with Ad-GFP or Ad-BMP9, cultured with or without GL-powder. Total RNA was isolated at 48 h and subjected to TqPCR analysis using gene-specific primers for mouse Osx, Opn, and Ocn. Gapdh was used as a reference gene. Reactions were done in triplicate. “**”, p < 0.05 when compared with the BMP9 group; “*”, p < 0.05 when compared with the GFP group.

Figure 6. Augmentation of BMP9-induced ectopic bone formation by GL-Powder. (A) μCT imaging analysis of ectopic bony masses. The retrieved bone masses from the iMEFs + BMP9 group and the iMEFs + BMP9 + GL-powder group were imaged using μCT followed by 3D reconstruction. No detectable masses were retrieved from the Ad-GFP-transduced iMEFs only group and iMEFs + GL-powder group (a). Representative images are shown. The average bone volumes for different groups were determined and analyzed using the Amira program (b). (B(a,b)) Hematoxylin and eosin staining of the retrieved bone masses. Representative images are shown. (B(c)) Quantitative measurement of the trabecular bone area was done using the ImageJ program. GL, GL-powder; MB, mineralized/mature bone matrix; MSCs, undifferentiated MSCs. “**”, p < 0.05 when compared with the BMP9 group.

However, the GL-powder can augment BMP9-induced osteogenesis, as evidenced by the increased ALP activity and matrix mineralization and the upregulated expression of osteogenic markers. Runx2, Osx, and ALP are expressed in the early phase, whereas Ocn and Opn are expressed in the middle or late phase. In our previous findings or the findings by other researchers, the expression of Ocn and Opn are usually dramatically increased after 7 days in vitro. However, the GL-powder was shown to induce robust Ocn and Opn expression even on the second day

with our results could partially explain the mechanism by which our 3D GL-scaffolds promote osteogenic differentiation of the stem cells. More importantly, our GL-scaffolds are inexpensive to produce and also highly scalable. Hence, the reported hybrid materials have high potential as a new class of tissue engineering scaffolds and as implantable medical devices. Nonetheless, we demonstrated that the GL-powder alone is not sufficient to induce osteogenic differentiation of MSCs, indicating that the 3D structure is beneficial for MSCs to form a bone. 15929



■

of culturing. Interestingly, the expression of early osteogenic marker ALP is downregulated in iMADs but upregulated in iMEFs. This may be because iMADs are more differentiated than iMEFs, so that when stimulated with BMP9, iMADs may be more likely to pass the early stage, resulting in a relatively low ALP activity. Alternatively, GL-powder may promote osteogenic differentiation of stem cells by accelerating the phase switch from the early to late stage, especially in MSCs that are more differentiated. Furthermore, we showed that the GL-powder enhanced the matrix mineralization of the stem cells stimulated with BMP9, which was also observed in single-sheet graphene.53 Moreover, they found that the promotional effect of graphene may be attributed to its ability to preconcentrate β-glycerolphosphate, underlying one of the possible mechanisms by which our GL-powder promotes matrix mineralization.53 To the best of our understanding, this work represents one of the first studies to assess the effect of graphene-based materials on BMP9-induced osteogenesis. BMPs belong to TGF-β superfamily and have great potentials to induce osteogenic differentiation of stem cells. Clinical applications of BMPs are currently focused on BMP2 and BMP7. Although Nayak reported that single-sheet graphene can induce osteogenic differentiation comparable to BMP2, it cannot enhance BMP2-induced osteogenic differentiation of MSCs.54 Our previous studies demonstrated that among the 14 types, BMP9 is one of the most potent to induce MSC osteogenic differentiation. Thus, a combination of BMP9 and GL hybrids may achieve the most robust bone formation.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.J.d.P.). *E-mail: [email protected] or [email protected]. Phone/Fax: 011-86-23-6382 2194 (Z.L.D.). ORCID

Juan J. de Pablo: 0000-0002-3526-516X Zhong-Liang Deng: 0000-0003-4831-1775 Author Contributions ††

Y.Z. and N.T.Q. contributed equally to the work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was supported by funding from the Center for Hierarchical Materials Design, CHiMAD (NIST award 70NANB14H012 to M.T. & J.J.D.P.), the National Natural Science Foundation of China (#grant no. 81672230 to Z.L.D.), the NIH grants (#AT004418, DE020140 to T.C.H. and R.R.R.), the US Department of Defense (OR130096 to J.M.W.), and the Scoliosis Research Society (T.C.H. and M.J.L.). Also, the reported work utilized The University of Chicago’s MRSEC Facilities, which were funded by the National Science Foundation (grant# NSF-DMR-1420709). Funding sources were not involved in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

■

5. CONCLUSIONS We used Laponite nanosheets to obtain gelatin-derived GL hybrids in 3D GL-scaffold and GL-powder forms and investigated their biomedical applications. The GL-scaffold can support homogenous attachment and long-term proliferation of MSCs. iMADs and iMEFs had an elongated spindlelike morphology on the scaffold, implying their differentiation states. All tested MSC lines formed well-mineralized regions on the scaffolds, indicating that the GL-scaffold can induce matrix mineralization. The GL-powder was shown to be biocompatible, increased the ALP activity of iMEFs, and promoted matrix mineralization of MSCs. A quantitative PCR analysis indicated that the GL-powder alone was shown to upregulate the expression of osteogenic markers (Runx2, Opn, and Ocn), and chondrogenic marker Sox9 to a certain extent, but not adipogenic marker PPARγ. The GL-powder was shown to synergistically upregulate BMP9-induced Osterix expression. Stem cell implantation experiments further demonstrated that the GL-powder was able to significantly enhance BMP9-induced ectopic bone formation from MSCs, indicating that the inclusion of GL-powder with BMP9-transduced MSCs can lead to a more robust bone formation in vivo. Taken together, our results demonstrate that GL-powder may synergistically promote BMP9-induced osteogenic differentiation of MSCs. Hence, the novel GL hybrid materials hold great potential for robust bone tissue engineering.

■

Research Article

REFERENCES

(1) Crane, G. M.; Ishaug, S. L.; Mikos, A. G. Bone Tissue Engineering. Nat. Med. 1995, 1, 1322−1324. (2) Szpalski, C.; Wetterau, M.; Barr, J.; Warren, S. M. Bone Tissue Engineering: Current Strategies and TechniquesPart I: Scaffolds. Tissue Eng., Part B 2012, 18, 246−257. (3) Karimi, F.; Qazvini, N. T.; Namivandi-Zangeneh, R. Fish gelatin/ Laponite Biohybrid Elastic Coacervates: A Complexation KineticsStructure Relationship Study. Int. J. Biol. Macromol. 2013, 61, 102−113. (4) Huang, E.; Bi, Y.; Jiang, W.; Luo, X.; Yang, K.; Gao, J.-L.; Gao, Y.; Luo, Q.; Shi, Q.; Kim, S. H.; Liu, X.; Li, M.; Hu, N.; Liu, H.; Cui, J.; Zhang, W.; Li, R.; Chen, X.; Shen, J.; Kong, Y.; Zhang, J.; Wang, J.; Luo, J.; He, B.-C.; Wang, H.; Reid, R. R.; Luu, H. H.; Haydon, R. C.; Yang, L.; He, T.-C. Conditionally Immortalized Mouse Embryonic Fibroblasts Retain Proliferative Activity without Compromising Multipotent Differentiation Potential. PLoS One 2012, 7, No. e32428. (5) Wang, N.; Zhang, W.; Cui, J.; Zhang, H.; Chen, X.; Li, R.; Wu, N.; Chen, X.; Wen, S.; Zhang, J.; Yin, L.; Deng, F.; Liao, Z.; Zhang, Z.; Zhang, Q.; Yan, Z.; Liu, W.; Ye, J.; Deng, Y.; Wang, Z.; Qiao, M.; Luu, H. H.; Haydon, R. C.; Shi, L. L.; Liang, H.; He, T.-C. The piggyBac Transposon-Mediated Expression of SV40 T Antigen Efficiently Immortalizes Mouse Embryonic Fibroblasts (MEFs). PLoS One 2014, 9, No. e97316. (6) Lu, S.; Wang, J.; Ye, J.; Zou, Y.; Zhu, Y.; Wei, Q.; Wang, X.; Tang, S.; Liu, H.; Fan, J.; Zhang, F.; Farina, E. M.; Mohammed, M. M.; Song, D.; Liao, J.; Huang, J.; Guo, D.; Lu, M.; Liu, F.; Liu, J.; Li, L.; Ma, C.; Hu, X.; Lee, M. J.; Reid, R. R.; Ameer, G. A.; Zhou, D.; He, T. Bone Morphogenetic Protein 9 (BMP9) Induces Effective Bone Formation from Reversibly Immortalized Multipotent Adipose-Derived (iMAD) Mesenchymal Stem Cells. Am. J. Transl. Res. 2016, 8, 3710−3730. (7) Shenaq, D. S.; Teven, C. M.; Seitz, I. A.; Rastegar, F.; Greives, M. R.; He, T. C.; Reid, R. R. Characterization of Reversibly Immortalized Calvarial Mesenchymal Progenitor Cells. J. Craniofac. Surg. 2015, 26, 1207−1213. (8) He, T.-C.; Zhou, S.; da Costa, L. T.; Yu, J.; Kinzler, K. W.; Vogelstein, B. A Simplified System for Generating Recombinant Adenoviruses. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2509−2514.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b00272. Primer3 Plus used to design TqPCR primers (usually 18− 20 mers, product sizes in the range of 120−200 bp) and sequences of primers (PDF) 15930


Research Article

ACS Applied Materials & Interfaces (9) Luo, J.; Deng, Z.-L.; Luo, X.; Tang, N.; Song, W.-X.; Chen, J.; Sharff, K. A.; Luu, H. H.; Haydon, R. C.; Kinzler, K. W.; Vogelstein, B.; He, T.-C. A Protocol for Rapid Generation of Recombinant Adenoviruses Using the AdEasy System. Nat. Protoc. 2007, 2, 1236− 1247. (10) Breyer, B.; Jiang, W.; Cheng, H.; Zhou, L.; Paul, R.; Feng, T.; He, T.-C. Adenoviral Vector-Mediated Gene Transfer for Human Gene Therapy. Curr. Gene Ther. 2001, 1, 149−162. (11) Cheng, H.; Jiang, W.; Phillips, F. M.; Haydon, R. C.; Peng, Y.; Zhou, L.; Luu, H. H.; An, N.; Breyer, B.; Vanichakarn, P.; Szatkowski, J. P.; Park, J. Y.; He, T.-C. Osteogenic Activity of the Fourteen Types of Human Bone Morphogenetic Proteins (BMPs). J. Bone Jt. Surg., Am. Vol. 2003, 85-A, 1544−1552. (12) Wu, N.; Zhang, H.; Deng, F.; Li, R.; Zhang, W.; Chen, X.; Wen, S.; Wang, N.; Zhang, J.; Yin, L.; Liao, Z.; Zhang, Z.; Zhang, Q.; Yan, Z.; Liu, W.; Wu, D.; Ye, J.; Deng, Y.; Yang, K.; Luu, H. H.; Haydon, R. C.; He, T.C. Overexpression of Ad5 Precursor Terminal Protein Accelerates Recombinant Adenovirus Packaging and Amplification in HEK-293 Packaging Cells. Gene Ther. 2014, 21, 629−637. (13) Zhang, H.; Wang, J.; Deng, F.; Huang, E.; Yan, Z.; Wang, Z.; Deng, Y.; Zhang, Q.; Zhang, Z.; Ye, J.; Qiao, M.; Li, R.; Wang, J.; Wei, Q.; Zhou, G.; Luu, H. H.; Haydon, R. C.; He, T.-C.; Deng, F. Canonical Wnt Signaling Acts Synergistically on BMP9-Induced Osteo/Odontoblastic Differentiation of Stem Cells of Dental Apical Papilla (SCAPs). Biomaterials 2015, 39, 145−154. (14) Huang, J.; Bi, Y.; Zhu, G.-H.; He, Y.; Su, Y.; He, B.-C.; Wang, Y.; Kang, Q.; Chen, L.; Zuo, G.-W.; Luo, Q.; Shi, Q.; Zhang, B.-Q.; Huang, A.; Zhou, L.; Feng, T.; Luu, H. H.; Haydon, R. C.; He, T.-C.; Tang, N. Retinoic Acid Signalling Induces the Differentiation of Mouse Fetal Liver-Derived Hepatic Progenitor Cells. Liver Int. 2009, 29, 1569−1581. (15) Tang, N.; Song, W.-X.; Luo, J.; Luo, X.; Chen, J.; Sharff, K. A.; Bi, Y.; He, B.-C.; Huang, J.-Y.; Zhu, G.-H.; Su, Y.-X.; Jiang, W.; Tang, M.; He, Y.; Wang, Y.; Chen, L.; Zuo, G.-W.; Shen, J.; Pan, X.; Reid, R. R.; Luu, H. H.; Haydon, R. C.; He, T.-C. BMP9-induced Osteogenic Differentiation of Mesenchymal Progenitors Requires Functional Canonical Wnt/β-Catenin Signaling. J. Cell. Mol. Med. 2009, 13, 2448−2464. (16) Liu, X.; Qin, J.; Luo, Q.; Bi, Y.; Zhu, G.; Jiang, W.; Kim, S. H.; Li, M.; Su, Y.; Nan, G.; Cui, J.; Zhang, W.; Li, R.; Chen, X.; Kong, Y.; Zhang, J.; Wang, J.; Rogers, M. R.; Zhang, H.; Shui, W.; Zhao, C.; Wang, N.; Liang, X.; Wu, N.; He, Y.; Luu, H. H.; Haydon, R. C.; Shi, L. L.; Li, T.; He, T.-C.; Li, M. Cross-Talk between EGF and BMP9 Signalling Pathways Regulates the Osteogenic Differentiation of Mesenchymal Stem Cells. J. Cell. Mol. Med. 2013, 17, 1160−1172. (17) Bi, Y.; Huang, J.; He, Y.; Zhu, G.-H.; Su, Y.; He, B.-C.; Luo, J.; Wang, Y.; Kang, Q.; Luo, Q.; Chen, L.; Zuo, G.-W.; Jiang, W.; Liu, B.; Shi, Q.; Tang, M.; Zhang, B.-Q.; Weng, Y.; Huang, A.; Zhou, L.; Feng, T.; Luu, H. H.; Haydon, R. C.; He, T.-C.; Tang, N. Wnt Antagonist SFRP3 Inhibits the Differentiation of Mouse Hepatic Progenitor Cells. J. Cell. Biochem. 2009, 108, 295−303. (18) Zhao, C.; Wu, N.; Deng, F.; Zhang, H.; Wang, N.; Zhang, W.; Chen, X.; Wen, S.; Zhang, J.; Yin, L.; Liao, Z.; Zhang, Z.; Zhang, Q.; Yan, Z.; Liu, W.; Wu, D.; Ye, J.; Deng, Y.; Zhou, G.; Luu, H. H.; Haydon, R. C.; Si, W.; He, T.-C. Adenovirus-Mediated Gene Transfer in Mesenchymal Stem Cells Can be Significantly Enhanced by the Cationic Polymer Polybrene. PLoS One 2014, 9, No. e92908. (19) Ye, J.; Wang, J.; Zhu, Y.; Wei, Q.; Wang, X.; Yang, J.; Tang, S.; Liu, H.; Fan, J.; Zhang, F.; Farina, E. M.; Mohammed, M. K.; Zou, Y.; Song, D.; Liao, J.; Huang, J.; Guo, D.; Lu, M.; Liu, F.; Liu, J.; Li, L.; Ma, C.; Hu, X.; Haydon, R. C.; Lee, M. J.; Reid, R. R.; Ameer, G. A.; Yang, L.; He, T.C. A Thermoresponsive Polydiolcitrate-Gelatin Scaffold and Delivery System Mediates Effective Bone Formation from BMP9-Transduced Mesenchymal Stem Cells. Biomed. Mater. 2016, 11, 025021. (20) Deng, Y.; Wang, Z.; Zhang, F.; Qiao, M.; Yan, Z.; Wei, Q.; Wang, J.; Liu, H.; Fan, J.; Zou, Y.; Liao, J.; Hu, X.; Chen, L.; Yu, X.; Haydon, R. C.; Luu, H. H.; Qi, H.; He, T.-C.; Zhang, J. A Blockade of IGF Signaling Sensitizes Human Ovarian Cancer Cells to the Anthelmintic Niclosamide-Induced Anti-Proliferative and Anticancer Activities. Cell. Physiol. Biochem. 2016, 39, 871−888.

(21) Deng, Y.; Zhang, J.; Wang, Z.; Yan, Z.; Qiao, M.; Ye, J.; Wei, Q.; Wang, J.; Wang, X.; Zhao, L.; Lu, S.; Tang, S.; Mohammed, M. K.; Liu, H.; Fan, J.; Zhang, F.; Zou, Y.; Liao, J.; Qi, H.; Haydon, R. C.; Luu, H. H.; He, T.-C.; Tang, L. Antibiotic Monensin Synergizes with EGFR Inhibitors and Oxaliplatin to Suppress the Proliferation of Human Ovarian Cancer Cells. Sci. Rep. 2015, 5, 17523. (22) Luo, X.; Chen, J.; Song, W.-X.; Tang, N.; Luo, J.; Deng, Z.-L.; Sharff, K. A.; He, G.; Bi, Y.; He, B.-C.; Bennett, E.; Huang, J.; Kang, Q.; Jiang, W.; Su, Y.; Zhu, G.-H.; Yin, H.; He, Y.; Wang, Y.; Souris, J. S.; Chen, L.; Zuo, G.-W.; Montag, A. G.; Reid, R. R.; Haydon, R. C.; Luu, H. H.; He, T.-C. Osteogenic BMPs Promote Tumor Growth of Human Osteosarcomas that Harbor Differentiation Defects. Lab. Invest. 2008, 88, 1264−1277. (23) Li, R.; Yan, Z.; Ye, J.; Huang, H.; Wang, Z.; Wei, Q.; Wang, J.; Zhao, L.; Lu, S.; Wang, X.; Tang, S.; Fan, J.; Zhang, F.; Zou, Y.; Song, D.; Liao, J.; Lu, M.; Liu, F.; Shi, L. L.; Athiviraham, A.; Lee, M. J.; He, T.-C.; Zhang, Z. The Prodomain-Containing BMP9 Produced from a Stable Line Effectively Regulates the Differentiation of Mesenchymal Stem Cells. Int. J. Med. Sci. 2016, 13, 8−18. (24) Deng, F.; Chen, X.; Liao, Z.; Yan, Z.; Wang, Z.; Deng, Y.; Zhang, Q.; Zhang, Z.; Ye, J.; Qiao, M.; Li, R.; Denduluri, S.; Wang, J.; Wei, Q.; Li, M.; Geng, N.; Zhao, L.; Zhou, G.; Zhang, P.; Luu, H. H.; Haydon, R. C.; Reid, R. R.; Yang, T.; He, T.-C. A Simplified and Versatile System for the Simultaneous Expression of Multiple siRNAs in Mammalian Cells Using Gibson DNA Assembly. PLoS One 2014, 9, No. e113064. (25) Chen, L.; Jiang, W.; Huang, J.; He, B.-C.; Zuo, G.-W.; Zhang, W.; Luo, Q.; Shi, Q.; Zhang, B.-Q.; Wagner, E. R.; Luo, J.; Tang, M.; Wietholt, C.; Luo, X.; Bi, Y.; Su, Y.; Liu, B.; Kim, S. H.; He, C. J.; Hu, Y.; Shen, J.; Rastegar, F.; Huang, E.; Gao, Y.; Gao, J.-L.; Zhou, J.-Z.; Reid, R. R.; Luu, H. H.; Haydon, R. C.; He, T.-C.; Deng, Z.-L. Insulin-Like Growth Factor 2 (IGF-2) Potentiates BMP-9-Induced Osteogenic Differentiation and Bone Formation. J. Bone Miner. Res. 2010, 25, 2447− 2459. (26) Luo, J.; Tang, M.; Huang, J.; He, B.-C.; Gao, J.-L.; Chen, L.; Zuo, G.-W.; Zhang, W.; Luo, Q.; Shi, Q.; Zhang, B.-Q.; Bi, Y.; Luo, X.; Jiang, W.; Su, Y.; Shen, J.; Kim, S. H.; Huang, E.; Gao, Y.; Zhou, J.-Z.; Yang, K.; Luu, H. H.; Pan, X.; Haydon, R. C.; Deng, Z.-L.; He, T.-C. TGFβ/BMP Type I Receptors ALK1 and ALK2 Are Essential for BMP9-Induced Osteogenic Signaling in Mesenchymal Stem Cells. J. Biol. Chem. 2010, 285, 29588−29598. (27) Untergasser, A.; Cutcutache, I.; Koressaar, T.; Ye, J.; Faircloth, B. C.; Remm, M.; Rozen, S. G. Primer3New Capabilities and Interfaces. Nucleic Acids Res. 2012, 40, No. e115. (28) Zhang, Q.; Wang, J.; Deng, F.; Yan, Z.; Xia, Y.; Wang, Z.; Ye, J.; Deng, Y.; Zhang, Z.; Qiao, M.; Li, R.; Denduluri, S. K.; Wei, Q.; Zhao, L.; Lu, S.; Wang, X.; Tang, S.; Liu, H.; Luu, H. H.; Haydon, R. C.; He, T.-C.; Jiang, L. TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR. PLoS One 2015, 10, No. e0132666. (29) Lamplot, J. D.; Liu, B.; Yin, L.; Zhang, W.; Wang, Z.; Luther, G.; Wagner, E.; Li, R.; Nan, G.; Shui, W.; Yan, Z.; Rames, R.; Deng, F.; Zhang, H.; Liao, Z.; Liu, W.; Zhang, J.; Zhang, Z.; Zhang, Q.; Ye, J.; Deng, Y.; Qiao, M.; Haydon, R. C.; Luu, H. H.; Angeles, J.; Shi, L. L.; He, T.-C.; Ho, S. H. Reversibly Immortalized Mouse Articular Chondrocytes Acquire Long-Term Proliferative Capability while Retaining Chondrogenic Phenotype. Cell Transplant. 2015, 24, 1053−1066. (30) Kang, Q.; Sun, M. H.; Cheng, H.; Peng, Y.; Montag, A. G.; Deyrup, A. T.; Jiang, W.; Luu, H. H.; Luo, J.; Szatkowski, J. P.; Vanichakarn, P.; Park, J. Y.; Li, Y.; Haydon, R. C.; He, T.-C. Characterization of the Distinct Orthotopic Bone-Forming Activity of 14 BMPs Using Recombinant Adenovirus-Mediated Gene Delivery. Gene Ther. 2004, 11, 1312−1320. (31) Li, Y.; Wagner, E. R.; Yan, Z.; Wang, Z.; Luther, G.; Jiang, W.; Ye, J.; Wei, Q.; Wang, J.; Zhao, L.; Lu, S.; Wang, X.; Mohammed, M. K.; Tang, S.; Liu, H.; Fan, J.; Zhang, F.; Zou, Y.; Song, D.; Liao, J.; Haydon, R. C.; Luu, H. H.; He, T.-C. The Calcium-Binding Protein S100A6 Accelerates Human Osteosarcoma Growth by Promoting Cell Proliferation and Inhibiting Osteogenic Differentiation. Cell. Physiol. Biochem. 2015, 37, 2375−2392. 15931


Research Article

ACS Applied Materials & Interfaces (32) Su, Y.; Wagner, E. R.; Luo, Q.; Huang, J.; Chen, L.; He, B.-C.; Zuo, G.-W.; Shi, Q.; Zhang, B.-Q.; Zhu, G.; Bi, Y.; Luo, J.; Luo, X.; Kim, S. H.; Shen, J.; Rastegar, F.; Huang, E.; Gao, Y.; Gao, J.-L.; Yang, K.; Wietholt, C.; Li, M.; Qin, J.; Haydon, R. C.; He, T.-C.; Luu, H. H. Insulin-Like Growth Factor Binding Protein 5 Suppresses Tumor Growth and Metastasis of Human Osteosarcoma. Oncogene 2011, 30, 3907−3917. (33) Wang, J.; Liao, J.; Zhang, F.; Song, D.; Lu, M.; Liu, J.; Wei, Q.; Tang, S.; Liu, H.; Fan, J.; Zou, Y.; Guo, D.; Huang, J.; Liu, F.; Ma, C.; Hu, X.; Li, L.; Qu, X.; Chen, L.; Weng, Y.; Lee, M. J.; He, T.-C.; Reid, R. R.; Zhang, J. NEL-Like Molecule-1 (Nell1) Is Regulated by Bone Morphogenetic Protein 9 (BMP9) and Potentiates BMP9-Induced Osteogenic Differentiation at the Expense of Adipogenesis in Mesenchymal Stem Cells. Cell. Physiol. Biochem. 2017, 41, 484−500. (34) Wang, J.; Zhang, H.; Zhang, W.; Huang, E.; Wang, N.; Wu, N.; Wen, S.; Chen, X.; Liao, Z.; Deng, F.; Yin, L.; Zhang, J.; Zhang, Q.; Yan, Z.; Liu, W.; Zhang, Z.; Ye, J.; Deng, Y.; Luu, H. H.; Haydon, R. C.; He, T.-C.; Deng, F. Bone Morphogenetic Protein-9 Effectively Induces Osteo/Odontoblastic Differentiation of the Reversibly Immortalized Stem Cells of Dental Apical Papilla. Stem Cells Dev 2014, 23, 1405− 1416. (35) Yan, Z.; Yin, L.; Wang, Z.; Ye, J.; Zhang, Z.; Li, R.; Denduluri, S. K.; Wang, J.; Wei, Q.; Zhao, L.; Lu, S.; Wang, X.; Tang, S.; Shi, L. L.; Lee, M. J.; He, T.-C.; Deng, Z.-L. A Novel Organ Culture Model of Mouse Intervertebral Disc Tissues. Cells Tissues Organs 2016, 201, 38−50. (36) Xavier, J. R.; Thakur, T.; Desai, P.; Jaiswal, M. K.; Sears, N.; Cosgriff-Hernandez, E.; Kaunas, R.; Gaharwar, A. K. Bioactive Nanoengineered Hydrogels for Bone Tissue Engineering: A GrowthFactor-Free Approach. ACS Nano 2015, 9, 3109−3118. (37) Childres, I.; Jauregui, L. A.; Park, W.; Cao, H.; Chen, Y. P. Raman Spectroscopy of Graphene and Related Materials. New Dev. Photon Mater. Res. 2013, 1, 1−20. (38) Rey, C.; Marsan, O.; Combes, C.; Drouet, C.; Grossin, D.; Sarda, S. Characterization of Calcium Phosphates Using Vibrational Spectroscopies. In Advances in Calcium Phosphate Biomaterials; Springer, 2014; pp 229−266. (39) Zhang, F.; Li, Y.; Zhang, H.; Huang, E.; Gao, L.; Luo, W.; Wei, Q.; Fan, J.; Song, D.; Liao, J.; Zou, Y.; Liu, F.; Liu, J.; Huang, J.; Guo, D.; Ma, C.; Hu, X.; Li, L.; Qu, X.; Chen, L.; Yu, X.; Zhang, Z.; Wu, T.; Luu, H. H.; Haydon, R. C.; Song, J.; He, T. C.; Ji, P. Anthelmintic Mebendazole Enhances Cisplatin’s Effect on Suppressing Cell Proliferation and Promotes Differentiation of Head and Neck Squamous Cell Carcinoma (HNSCC). Oncotarget 2017, 8, 12968−12982. (40) Denduluri, S. K.; Scott, B.; Lamplot, J. D.; Yin, L.; Yan, Z.; Wang, Z.; Ye, J.; Wang, J.; Wei, Q.; Mohammed, M. K.; Haydon, R. C.; Kang, R. W.; He, T.-C.; Athiviraham, A.; Ho, S. H.; Shi, L. L. Immortalized Mouse Achilles Tenocytes Demonstrate Long-Term Proliferative Capacity while Retaining Tenogenic Properties. Tissue Eng., Part C 2016, 22, 280−289. (41) Peng, Y.; Kang, Q.; Cheng, H.; Li, X.; Sun, M. H.; Jiang, W.; Luu, H. H.; Park, J. Y.; Haydon, R. C.; He, T.-C. Transcriptional Characterization of Bone Morphogenetic Proteins (BMPs)-mediated Osteogenic Signaling. J. Cell. Biochem. 2003, 90, 1149−1165. (42) Peng, Y.; Kang, Q.; Luo, Q.; Jiang, W.; Si, W.; Liu, B. A.; Luu, H. H.; Park, J. K.; Li, X.; Luo, J.; Montag, A. G.; Haydon, R. C.; He, T.-C. Inhibitor of DNA Binding/differentiation Helix-Loop-Helix Proteins Mediate Bone Morphogenetic Protein-induced Osteoblast Differentiation of Mesenchymal Stem Cells. J. Biol. Chem. 2004, 279, 32941−32949. (43) Sharff, K. A.; Song, W.-X.; Luo, X.; Tang, N.; Luo, J.; Chen, J.; Bi, Y.; He, B.-C.; Huang, J.; Li, X.; Jiang, W.; Zhu, G.-H.; Su, Y.; He, Y.; Shen, J.; Wang, Y.; Chen, L.; Zuo, G.-W.; Liu, B.; Pan, X.; Reid, R. R.; Luu, H. H.; Haydon, R. C.; He, T.-C. Hey1 Basic Helix-Loop-Helix Protein Plays an Important Role in Mediating BMP9-induced Osteogenic Differentiation of Mesenchymal Progenitor Cells. J. Biol. Chem. 2009, 284, 649−659. (44) Ruiz-Hitzky, E.; Sobral, M. M. C.; Gómez-Avilky, A.; Nunes, C.; Ruiz-García, C.; Ferreira, P.; Aranda, P. Clay-Graphene Nanoplatelets Functional Conducting Composites. Adv. Funct. Mater. 2016, 26, 7394− 7405.

(45) Zhang, X.; Jiao, Y.; Sun, L.; Wang, L.; Wu, A.; Yan, H.; Meng, M.; Tian, C.; Jiang, B.; Fu, H. GO-Induced Assembly of Gelatin toward Stacked Layer-Like Porous Carbon for Advanced Supercapacitors. Nanoscale 2016, 8, 2418−2427. (46) Nam, G.; Park, J.; Kim, S. T.; Shin, D.-B.; Park, N.; Kim, Y.; Lee, J.S.; Cho, J. Metal-free Ketjenblack Incorporated Nitrogen-doped Carbon Sheets Derived from Gelatin as Oxygen Reduction Catalysts. Nano Lett. 2014, 14, 1870−1876. (47) Guan, Z.; Liu, H.; Xu, B.; Hao, X.; Wang, Z.; Chen, L. Gelatinpyrolyzed Mesoporous Carbon as a High-performance Sodium-Storage Material. J. Mater. Chem. A 2015, 3, 7849−7854. (48) Fan, X.; Yu, C.; Yang, J.; Ling, Z.; Hu, C.; Zhang, M.; Qiu, J. A Layered-Nanospace-Confinement Strategy for the Synthesis of TwoDimensional Porous Carbon Nanosheets for High-Rate Performance Supercapacitors. Adv. Energy Mater. 2015, 5, 1401761. (49) Crowder, S. W.; Prasai, D.; Rath, R.; Balikov, D. A.; Bae, H.; Bolotin, K. I.; Sung, H.-J. Three-dimensional Graphene Foams Promote Osteogenic Differentiation of Human Mesenchymal Stem Cells. Nanoscale 2013, 5, 4171−4176. (50) Oh, S.; Brammer, K. S.; Li, Y. S. J.; Teng, D.; Engler, A. J.; Chien, S.; Jin, S. Stem Cell Fate Dictated Solely by Altered Nanotube Dimension. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 2130−2135. (51) Kim, J.; Kim, Y.-R.; Kim, Y.; Lim, K. T.; Seonwoo, H.; Park, S.; Cho, S.-P.; Hong, B. H.; Choung, P.-H.; Chung, T. D.; Choung, Y.-H.; Chung, J. H. Graphene-incorporated Chitosan Substrata for Adhesion and Differentiation of Human Mesenchymal Stem Cells. J. Mater. Chem. B 2013, 1, 933−938. (52) Akhavan, O.; Ghaderi, E.; Shahsavar, M. Graphene Nanogrids for Selective and Fast Osteogenic Differentiation of Human Mesenchymal Stem Cells. Carbon 2013, 59, 200−211. (53) Lee, W. C.; Lim, C. H. Y. X.; Shi, H.; Tang, L. A. L.; Wang, Y.; Lim, C. T.; Loh, K. P. Origin of Enhanced Stem Cell Growth and Differentiation on Graphene and Graphene Oxide. ACS Nano 2011, 5, 7334−7341. (54) Nayak, T. R.; Andersen, H.; Makam, V. S.; Khaw, C.; Bae, S.; Xu, X.; Ee, P.-L. R.; Ahn, J.-H.; Hong, B. H.; Pastorin, G.; Ö zyilmaz, B. Graphene for Controlled and Accelerated Osteogenic Differentiation of Human Mesenchymal Stem Cells. ACS Nano 2011, 5, 4670−4678.

15932


Gelatin-Derived GrapheneâSilicate Hybrid Materials Are

Recommend Documents

Gelatin-Derived GrapheneâSilicate Hybrid Materials Are