Fabrication, Characterization, and Biocompatibility of Polymer Cored

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Fabrication, Characterization and Biocompatibility of Polymer Cored Reduced Graphene Oxide Nanofibers Lin Jin, Dingcai Wu, Shreyas Kuddannaya, Yilei Zhang , and Zhenling Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00243 • Publication Date (Web): 02 Feb 2016 Downloaded from http://pubs.acs.org on February 6, 2016

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ACS Applied Materials & Interfaces

Fabrication, Characterization and Biocompatibility of Polymer Cored Reduced Graphene Oxide Nanofibers Lin Jin,†,‡ Dingcai Wu, §* Shreyas Kuddannaya, ‡ Yilei Zhang, ‡* Zhenling Wang†* †

The Key Laboratory of Rare Earth Functional Materials and Applications, Zhoukou

Normal University, Zhoukou 466001, P. R. China ‡

School of Mechanical & Aerospace Engineering, Nanyang Technological University,

50 Nanyang Avenue, Singapore 639798, Singapore Materials Science §

Institute, PCFM Lab and DSAPM Lab, School of Chemistry and Chemical

Engineering, Sun Yat-sen University, Guangzhou 510275, P. R. China ABSTRACT: Graphene nanofibers have shown a promising potential across a wide spectrum of areas, including biology, energy and environment. However, fabrication of graphene nanofibers remains a challenging issue due to the broad size distribution and extremely poor solubility of graphene. Herein, we report a facile yet efficient approach for fabricating a novel class of polymer core-reduced graphene oxide shell nanofiber mat (RGO-CSNFM) by direct heat-driven self-assembly of graphene oxide sheets onto the surface of electrospun polymeric nanofibers without any requirement of

surface

treatment.

Thus-prepared

RGO-CSNFM

demonstrated

excellent

mechanical, electrical and biocompatible properties. RGO-CSNFM also promoted a higher cell anchorage and proliferation of human bone marrow mesenchymal stem cells (hMSCs) compared to the free-standing RGO film without the nanoscale fibrous structure. Further, cell viability of hMSCs was comparable to that on the tissue culture plates (TCPs) with a distinctive healthy morphology, indicating that the nanoscale fibrous architecture plays a critically constructive role in supporting cellular activities. In addition, the RGO-CSNFM exhibited excellent electrical conductivity, making them an ideal candidate for conductive cell culture, bio-sensing and tissue engineering applications. These findings could provide a new benchmark for preparing well-defined graphene-based nanomaterial configurations and interfaces for bio-medical applications. KEYWORDS: graphene, electrospinning, nanofibers, hMSCs, tissue engineering

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1. INTRODUCTION Graphene is a two-dimensional monolayer of sp2-bonded carbon atoms with extraordinary electrical and mechanical properties, which has attracted tremendous research interest in recent years.1-11 Various techniques, such as electrospining, self-assembly, chemical vapor deposition and doping, have been employed to produce graphene-based materials in various configurations including graphene coated nanoparticles, graphene composite fibers, graphene membranes and papers for applications in biomedicine, energy storage, electronic devices, sensors and others.12-28 Over the years, the properties of graphene based materials (E.g, nanofibers) have been exploited in the field of tissue engineering; for example, they could mimic natural extracellular matrix (ECM) and govern specific cellular responses,29 including cell adhesion, migration, proliferation and differentiation. Electrospinning technique has been widely used to design nano-fibrous platforms due to its simplicity, versatility, and ease of scaling up. Morever, the morphology of electrospun nanofibers can be controlled easily by changing the electrospinning conditions including polymer concentration, feed rate, collector distance, and applied voltage.30-35 Hereby, in the view of utilizing the versatile properties of graphene in nano-fibrous configuration, previous reports have shown that graphene/polymer composite nanofiber membrane could be prepared by electrospinning and could be used as an optical element in fiber lasers.12 However, in such graphene composite nanofibers, the extraordinary properties and reactivity of graphene were seriously limited by low content of graphene oxide (GO) or reduced graphene oxide (RGO). Further, with a poor control over the graphene sheet organization, the disordered graphene sheet architecture within the composite nanofibers resulted in a degradation of its electrical and mechanical properties. To overcome these obstacles, a novel spinning process to fabricate neat “core-shell” structured graphene aerogel fibers was developed.24 Owing to certain fabrication process constraints, the dimension of the produced fibers was measured to be larger than tens of micrometers,23-26 which made them unsuitable for tissue engineering applications involving the mimicking of critical cell-material interactions and ECM architectures at nano-scale level which are shown to impact cell anchorage, survival and differentiation. 36,37 Herein, we propose a facile yet efficient electrospinning technique to develop a


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class of novel polymer core-reduced graphene oxide shell nanofiber mat (i.e., RGO core-shell nanofiber mat, RGO-CSNFM). As shown in Figure 1, the fabrication procedure involves three steps: Firstly, polymer nanofiber mat (PNFM) is prepared by electrospinning into the GO solution. Secondly, polymer core-graphene oxide shell nanofiber mat (i.e., GO core-shell nanofiber mat, GO-CSNFM) is formed by heat-driven self-assembly of GO sheets onto the surface of electrospun polymer nanofibers of PNFM in solution without any requirement of surface treatment. Thirdly, RGO-CSNFM is fromed by chemical reduction of GO core-shell nanofibers. Our method is easy to scale up, eco-friendly, and capable of constructing free-standing RGO-CSNFM with excellent electrical and mechanical properties. More importantly, compared to the conventional flat RGO film, as-prepared RGO-CSNFM promotes constructive cell-material interaction with a significantly improved adhesion and viability of hMSCs on their porous nanofibrous matrix environments. Considering the unusual integration of the unique fibrous nanostructure and the outstanding electrical, mechanical and biocompatible properties, we hope that the RGO-CSNFM could provide

promising opportunities to propel the

development of emerging

biotechnological areas, such as electrically active substrates/scaffolds for tissue engineering, biosensors, and controlled and sustained drug release. 2. EXPERIMENTAL SECTION 2.1 Preparation of RGO-CSNFM In this study, GO was prepared using natural graphite by employing a previously reported method by Kovtyukhova et al.38, 39 In the experiments, ultrapure Milli-Q water was used during every step as aqueous medium. Moreover, GO sheets were obtained from the dispersion of 1mg/mL GO aqueous solution through a ultrasonication method (at a frequency of 40 kHz and a output power of 100W), and then the obtained brown colored dispersion was centrifugated for 0.5 h at 3000 rpm to remove possible traces of un-exfoliated graphene oxide (usually nothing present). The polymeric nanofibrous core (PNFM) was fabricated by an optimized electrospinning process. Briefly, 10.0% PVC and 3.0% PLGA were dissolved in dichloromethane (THF)/N, N-dimethyl-formamide (DMF) (v: v = 8: 2). The resulting mixture was constantly stirred to form a clear and homogenous mixture solution. Subsequently, the obtained mixture solution was poured into a syringe capped with a



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0.22 gauge blunt-tripped needle nozzle. The syringe was fixed into a syringe pump (Langer CO., Baoding, China) adjusted with a flow speed of 1.0 mL/h. The PVC/PLGA nanofibers were then produced by electrospinning into the GO aqueous solution (0.10mg/mL). The syringe needle was placed 20 cm from the surface of GO solution and attached to the positive terminal of a high voltage DC power supply (Dongwen High Voltage, Tianjing, China). Finally, a voltage of 14 kV was applied from the needle opening. The stable hydrosol of graphene oxide and nanofibers was heated to 353 K for various short cycles (5 min, 30 min, 90 min, 2 h) in a thermostatic water bath, and then the obtained polymer and GO core-shell nanofiber mat was dried at 80 ºC for 24 h, during which a smooth and condensed thin graphene oxide layer was formed rapidly on the nanofibers. To improve its electrical conductivity, the obtained GO-CSNFM was dipped in a HI aqueous solution (55%) at 100 ºC for 1 h, and then RGO-CSNFM was obtained after washing repeatedly with ethanol to remove the residual HI. 2.2 Structural and Chemical Characterization For morphological analysis, PNFM and GO-CSNFM were mounted on the conductive adhesive surface, and then the samples were sputter-coated by gold and palladium. A Hitachi S-4800 equipment at an acceleration voltage of 10 kV was used to observe the images of the obtained mats. The morphology of the obtained GO sheets was assessed by a CypherTM atomic force microscope (AFM) in tapping mode and operated at the ambient conditions. Transmission electron microscope (TEM) was performed on JEOL-2100F with a field emission gun operating at 200 kV. The mechanical properties of RGO-CSNFM were evaluated according to our previously reported method32 using a micro-tensile testing system (Sans-GB T528, Shen Zhen, China) at a speed of 1mm/min. The conductivities were determined using a Van Der Pauw four-point setup at room temperature (HL550PC, Accent Optical, UK). The chemical compositions were examined by Raman spectroscopy (RIGAKU Co., Japan) obtained under a green laser (532 nm) with a power of 2 mW, FTIR equipped with an ATR model (Nexus, Thermo, Scientific, USA) and XRD (Bruker D8 Advance, Germany) with Cu-Kα radiation (λ = 0.15406 nm). 2. 3 Biocompatibility Analysis Human bone marrow mesenchymal stem cells (hMSCs) were obtianed from side


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population of human PLC/PRF/5 cell line (ATCC, Manassas, VA, USA) with flow cytometry of Hoechst 33342 (Invitrogen, Carlsbad, CA, USA). And then, hMSCs were kept in the complete dulbecco's modified eagle medium (DMEM), and used for the following experiments within 2 h. The RGO-CSNFM was cut into 16 mm discs and placed into 24-well plates as described in our previous work,31,33 and the samples were sterilized by soaking them in a phosphate buffered saline (PBS)-ethanol solution (70% ethanol and 30% PBS) for 6 h, followed by washing several times using PBS solution. The samples were immersed in DMEM for 12 h for protein adsorption. Before cell seeding, excess cell culture media for soaking scaffolds were removed and the cells were seeded according to our previous work.33 Briefly, hMSCs were seeded onto the RGO-CSNFM, RGO film and TCPs at a cell density of 1.6 × 104 cells/well with 0.5 mL DMEM supplemented with 10% (v/v) FBS. Cells were incubated under a normal humidified condition (37ºC, 5% CO2). To evaluate cell viability, cellular nuclei and cytoskeleton were visualized using Hoechst 33258 (5µg/mL, blue, Sigma-Aldrich) and Alexa Fluor 546®phalloidin (5µg/mL, red, Sigma-Aldrich)

respectively by incubation in the culture medium for

20 min. Then cells were fixed using 3.7% paraformaldehyde for 30 min, and images were taken under a Leica TCS-SP2 Confocal Microscope (Leica, Germany) and analyzed using TCS Leica Software 2.61. Cellular morphology on the RGO-CSNFM, RGO film and TCPs was visualized by SEM images. The cells on the substrate were fixed using 3% glutaraldehyde for 12 h and dehydrated using ethanol/water solution with various concentrations for 30 min.31 Cells were dried in air overnight. The above samples were sputter-coated with gold and palladium on the sample for 10 nm in thickness using a sputter coater and images were taken by SEM. Cellular adherence and proliferation were quantified at the specified time points by using a DNA analysis method.40, 41 Briefly, at each time point, PicoGreen® DNA quantification (Quant-It Picogreen, P7589, Invitrogen) reagent was incubated with each lysate for 5 min to measure the DNA content of the samples according to the manufacturer’s instructions. Fluorescence of the samples was tested at 485/535 nm using a Victor3 multilabel fluorescence plate reader (PerkinElmer, USA), and the cell number was calculated from the absorbance standard of the known cell concentration.



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3. RESULTS AND DISCUSSION The fabrication procedure of RGO-CSNFM, including electrospinning, self-assembly and chemical reduction is shown in Figure 1. For electrospinning, a suitable polymer should be selected based on the application requirements. PLGA is a hydrophilic polymer with a good biocompatibility, and PVC is a readily available cost-effective synthetic polymer with stable chemical and physical properties. In this work,

a

mixed

solution

of

poly

(lactic-co-glycolic

acid)

(PLGA)

and

polyvinylchloride (PVC) (10.0% PVC and 3.0% PLGA) was used to fabricate the core of electrospun nanofibers. The PLGA/PVC blended electrospun nanofiber possesses good physicochemical properties and is hydrophilic (Figure 2), and hence it is convenient for subsequent self-assembly of GO sheets in aqueous solutions. The PLGA/PVC solution was directly electrospun into a well-dispersed aqueous solution of GO (0.10 mg/mL) to form PLGA/PVC nanofibers. The GO sheets in the aqueous solution present irregular shapes and lateral dimensions, the size of them rang from several hundred nanometers to several micrometers (Figure S1). The average thickness of the GO sheets is around 1nm, slightly thicker than the monolayer graphene sheet (0.776 nm) due to the oxygen functional groups bearing on the basal planes and edges of the hydrophilic GO sheets.42-45 Subsequently, the GO solution with the PLGA/PVC nanofibers was heated to 353 K for 30 min in a thermostatic water bath, leading to the in-situ self-assembly of GO sheets onto the surface of the PLGA/PVC nanofibers to form a smooth and condensed GO nanoshell. After taking out from the aqueous solution and drying, GO-CSNFM was obtained, and then was chemically reduced using HI aqueous solution to obtain RGO-CSNFM. The yellow color of GO-CSNFM (Figure 3A) changes to black with typical metallic luster for RGO-CSNFM (Figure 3B). The diameter of the native PVC/PLGA nanofibers is 550±30nm and their surface is smooth and uniform (Figure 3C). However, after the self-assembly and chemical reduction, the diameter of the nanofibers of RGO-CSNFM increases to 580±30nm, and the nanofiber surface presents a nanoscale morphology due to the wrapping of graphene oxide sheets (Figure 3D and Figure 3E) around the polymeric core. The polymer nanofiber core-RGO shell nanostructure of RGO-CSNFM could be clearly observed after peeling off graphene layer from the RGO wrapped nanofibers using ultrasonic cleaner (Figure 3E, Figure 4 and Figure S2). Furthermore, the TEM image of RGO-CSNFM (Figure 3F) also clearly


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demonstrates the core-shell structure. These results confirm that the RGO nano-shell can be retained well during the chemical reduction process. The heating time of nanofibers in the GO solution is critical and optimal: too short or too long heating time could not lead to the formation of the desired core-shell structures. After 5 min of heating, the GO sheets begin to attach on the nanofibers (Figure 5A), and most of the GO sheets have already partially wrapped around the surface of nanofibers (Figure 5B). After 1 h, the GO sheets accumulate together and start to form a well spread thin GO film over most of the surface area of the underlying nanofibers instead of wrapping around the surface of individual nanofibers (Figure 5C). After 2 h, a consistent GO film was formed with upon underlying embedded nanofibers (Figure 5D) covering the whole surface area of the nanofibers. Therefore, 30 min is the optimal heating time for forming the well-defined polymer nanofiber core-RGO shell structure (Figure 4 and 3F). Chemical composition of the PLGA/PVC nanofibers, the GO wrapped nanofibers and the RGO wrapped nanofibers were characterized using Raman spectroscopy, XRD pattern and FTIR spectrum. After the self-assembly of GO sheets, GO characteristic Raman peaks (D peaks ~1590 cm-1 and G peaks ~1320 cm-1) are clearly shown on GO-CSNFM (Figure 6A). After chemical reduction, the 2D peak (2647 cm-1) increases obviously for RGO-CSNFM. These Raman peaks for RGO-CSNFM are similar to those found in the pristine graphite, confirming the successful reduction of GO-CSNFM using the HI acid.46-49 XRD pattern of GO-CSNFM indicates that an appearance of a strong reflection with peak at 2θ = 9.79°, which could be attributed to the (002) reflection of GO (Figure 6B). After the chemical reduction, the (002) reflection peak shifts from 9.79° to 24.08°, likely due to the elimination of the oxygen containing groups in the lower d-spacing. FTIR spectrum (Figure 6C) confirms that the dominant oxygen-containing groups, including -OH and C=O stretching of carboxylic acid and ester groups, have almost been removed completely after the chemical reduction.38, 50 These results combined together with the SEM image (Figure 4) and TEM image (Figure 3F) indicate that a successful construction of the polymer nanofiber core-shell nanostructure by the union of self-assembly and chemical reduction. Mechanical properties of PNFM, GO-CSNFM and RGO-CSNFM were characterized under tensile loading at room temperature (Figure 6D) with typical fracture strengths of 1.3±0.1 MPa, 4.0±0.13 MPa and 5.19±0.22 MPa, at ultimate



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elongations of 8.17±0.20%, 2.74±0.13% and 4.23±0.16%, respectively. The significant increase of fracture strength from PNFM to GO-CSNFM is due to the wrapping of GO sheets, while the increase of fracture strength from, GO-CSNFM to RGO-CSNFM could be attributed to the more compact stacking of graphene sheets after the chemical reduction of GO sheets. Therefore, the unique “core-shell” structure with aligned graphene building blocks, which is likely responsible for enhancing the strength of the RGO wrapped graphene nanofibers. These results clearly show that the obtained RGO-CSNFM exhibits advanced mechanical performance. The electrical conductivity of the RGO-CSNFM was characterized by four-probe method, and the result shows that the RGO-CSNFM becomes conductive and attains an electrical conductivity around 10.0 S/cm after reduction, which is much higher compared to the RGO composite nanofibers in the previous reports.13 Furthermore, the electrochemical performance of RGO-CSNFM was characterized in the PBS solution (0.1 M, pH=7.4) by cyclic voltammetry (CV) using a three-electrode system. RGO-CSNFM was used as a working electrode, and a platinum wire was used as a counter electrode against an Ag/AgCl reference electrode. RGO-CSNFM with a size of 2 cm × 2 cm was immersed in PBS, and the voltammograms were recorded with a scan rate of 50 mV/s. The CV curves in Figure 6E indicate that RGO-CSNFM exhibits a higher charge carrying capacity for a given voltage and a much better conductivity compared to GO-CSNFM and RGO- CSNFM composite nanofibers.41 The excellent electrical properity of RGO-CSNFM could be attribuated to the large surface area of RGO-CSNFM as well as the large number of π-π conjugated bonds generated near perfect spatial continuity over the entire surface of the RGO shell layer, permitting efficient electron transfer. Thus, RGO-CSNFM could provide an appropriate range of current for electrical stimulation required in certain specialized cell culture system. 51,52 Hydrophilic surfaces and reactive interfaces are desirable for biocompatibility of a biomaterial. In Figure 6F, static water contact angles measured on RGO-CSNFM and compared with a flat RGO film indicate that RGO-CSNFM is relatively hydrophilic (56.35±2.6°) compared to the RGO film (92.94±3.2°). Which was most likely due to two main reasons: one is the capillary effects at nanoscale fibrous structure; another is the PLGA as the core section also promoted the hydrophilic property of core-shell reduced graphene oxide nanofibers. In order to evaluate the biocompatibility of RGO-CSNFM, hMSCs were cultured on the RGO-CSNFM, and RGO film. Tissue


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culture plates (TCPs) were used as the control samples. Cell adherence through focal adhesion and morphology on these different substrates were assessed a by confocal fluorescent microscopy and SEM after 72 h of culture. Figure 7A shows the cystoskeletal and nuclear organization and orientation on different substrates. The hMSCs cultured on RGO-CSNFM display directional spreading of F-actin filaments which further tend to spread at the the cellular extensions along the nanofibers. In comparison, hMSCs on the TCPs show a round shape, because of an isotropic surface allowing hMSCs to spread in all directions, and hMSCs on the RGO film show poor distribution across the surface and cell aggregation which could be attributed to lower anchorage of cells on the relatively hydrophobic surface of RGO films. These results obviously indicate that the nanoscale fibrous architecture of RGO-CSNFM can effectively guide the focal adhesion of F-actin with attachment and alignment along nanofibers, which is very beneficial to the cell retention, survival, proliferation and differentiation.53,54 The detailed morphology of hMSCs cultured on TCPs, RGO film and RGO-CSNFM was studied by SEM after 72 h of culture. The hMSCs on the TCPs display a large number of cellular pesudopods and spread randomly on the isotropic surface of TCPs (Figure 7B). In contrast, hMSCs on RGO-CSNFM grow along the stretched direction of the nanofiber and display a firmly integrated cell-fiber constructs with a healthy anchorage. On the contrary, hMSCs on the RGO film could not be closely adhered to each other and only shrink tightly together, which could be attributed to the poor biocompatibility of the surface. A higher cell area with well spread morphology along all the focal adhesion points of hMSCs indicates healthy anchorage and survival of cells.55 From Figure 7 (A and B), it is clearly indicated that hMSCs on RGO-CSNFM surfaces exhibit higher surface area and good cell-cell contact compared to RGO films which have a smaller surface area with clumped cell distribution which could eventually lead to cell death. These findings reveals that the nanoscale size and unique RGO shell structure of the nanofibers for the RGO-CSNFM are much more helpful for the maintenance of cell activity and growth when compared to the RGO film due to a constructive synergistic interaction between cells and RGO core-shell nanofibers. During the 7 days culture period, cellular attachment and proliferation were qualitatively evaluated by DNA assay (Figure 8). After 6 h of culture, the cellular attachment of hMSCs on TCPs, RGO film and RGO-CSNFM is 93.3%, 84% and



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92%, respectively. The amount of attached cells on RGO-CSNFM and TCPs are close to each other, and their numbers are much higher than that on the RGO film. After 3 days of culture, the cell number on RGO-CSNFM (2.96×104) is slightly more than that on TCPs (2.945×104), and much higher than that on the RGO film (2.13×104). After 5 days of culture, the proliferation of cells on RGO-CSNFM shows a notable increase. The number of cells on RGO-CSNFM (4.52×104) is slightly less as compared to that on TCPs (4.96×104), while the cell number on the RGO film is still very low (3.6×104). After 7 days of culture, hMSCs on RGO-CSNFM (6.87×104) display a very similar cellular proliferation rate to that on TCPs (6.712×104), which is much better than that on RGO film (4.65×104). These results demonstrate that RGO-CSNFM can provide a suitable cellular microenvironment for promoting hMSCs proliferation and activity, because their nanoscale size and ECM-like fibrous structure help to counteract toxicity of the graphene film, making the obtained RGO-CSNFM a promising candidate in tissue engineering and biomedical devices, particularly in those systems where requiring high electrical conductivity and/or high mechanical strength. 4. CONCLUSION In conclusion, we successfully developed a novel class of high-performance RGO-CSNFM by the electrospinning with a heat-driven self-assembly and reduction process. The obtained RGO-CSNFM demonstrates highly advantageous nano-fibrous structure with excellent mechanical, electrical and hydrophilic properties. The in vitro cell culture experiments indicate that RGO-CSNFM could effectively enhance cells adhesion, growth and proliferation of hMSCs, thereby showing a potential for tissue engineering and further regenerative studies. Moreover, RGO core-shell nanofibrous architecture of the RGO-CSNFM proved highly advantageous for the interaction between the cells and the nanofibers, and has a much better cellular response when compared to the RGO film, thereby mimicking the native cellular micro-environments. We further believe that RGO-CSNFM might have potential application as substrates for engineered bio-scaffold with electrical stimulation and aid in development of smart biosensor interfaces which could be tuned to collect physiological signals. AUTHOR INFORMATION

Dr. L. Jin, Prof. Z. L. Wang*


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†

The Key Laboratory of Rare Earth Functional Materials and Applications, Zhoukou

Normal University, Zhoukou 466001, P. R. China E-mail: [email protected] Fax: 86394-8178518; Tel: 86394-8178996 S. Kuddannaya, Prof. Y. L. Zhang* ‡

School of Mechanical & Aerospace Engineering, Nanyang Technological University,

50 Nanyang Avenue, Singapore 639798, Singapore Materials Science E-mail: [email protected] Prof. D. C. Wu* §

Institute, PCFM Lab and DSAPM Lab, School of Chemistry and Chemical

Engineering, Sun Yat-sen University, Guangzhou 510275, P. R. China E-mail: [email protected] Supporting Information Supporting Information is available: This material is available free of charge via the Internet at http://pubs.acs.org. Typical AFM image and the corresponding height profile of the as-prepared GO sheets (Figure S1), High magnification SEM image of nanofibers after peeled off using ultrasonication for 5s (Figure S2). ACKNOWLEDGEMENTS This research was support by the National Natural Science Foundation of China (Grant No: 21404124, 51572303). Z. Wang acknowledges the project of Innovation Scientists and Technicians Troop Construction Projects of Henan Province (No: 2013259), Program for Innovative Research Team (in Science and Technology) in University of Henan Province (14IRTSTHN009), Excellent Youth Foundation of He’nan Scientific Committee (134100510018) and the project of Henan Province Key Discipline of Applied Chemistry (No:201218692). L. Jin acknowledges the project of Science and Technology Program of Henan Province (162102310591). Y. Zhang thanks Tier-1 Academic Research Funds from the Singapore Ministry of Education (RGT 30/13, RGC 6/13 and RGC 1/14), the A*STAR AOP project (1223600005) and the A*STAR Industrial Robotics Programme (1225100007). REFERENCES



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Figure 1 Schematic diagram of the RGO core-shell nanofiber mat fabrication process involving electrospinning, heat-driven self-assembly and reduction steps.



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Figure 2 Water droplet contact angle on the PVC (64.65±2.6°) and PVC/PLAG nanofiber surfaces (48.7±3.5°).


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Figure 3 Optical photos of (A) GO-CSFM and (B) RGO-CSFM; SEM images of (C) PNFM and (D) RGO-CSFM; (E) SEM image of nanofibers in RGO-CSFM after a moderate peeling off the core induced by ultrasonication for 5s; (F) TEM image of a single core-shell nanofiber.



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Figure 4 SEM image of nanofibers in RGO-CSFM after a peel off induced by ultrasonication.


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Figure 5 SEM images of the GO wrapped along the surface of PNFM in GO solution after various heating times: (A, B) 5 min, (C) 1 h and (D) 2 h.



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Figure 6 (A) Raman spectroscopy of PNFM, GO-CSNFM and RGO-CSNFM; (B) XRD patterns and (C) FTIR spectrum of GO-CSNFM and RGO-CSNFM; (D) Mechanical tensile stress and strain of PNFM, GO-CSNFM and RGO-CSNFM; (E) Cyclic voltammetry (CV) of GO-CSNFM and RGO-CSNFM; (F) Water droplet contact angle of the RGO film (92.94±3.2°) and RGO-CSNFM (56.35±2.6°).


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Figure 7 (A) Fluorescent images of hMSCs cultured on TCPs (left), RGO film (middle) and RGO-CSNFM (right) at 72h. Red: F-actin (Alexa Fluor 546®phalloidin); blue: nuclei (Hoechst 33258). (B) SEM images of hMSCs on various substrates: TCPs (left), RGO film (middle) and RGO-CSNFM (right).



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Figure 8 Evaluation of attachment and proliferation of hMSCs cultured on different substrates in various incubation periods by the PicoGreen dsDNA assay. Data are represented as mean ±SD, n = 3.


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Fabrication, Characterization, and Biocompatibility of Polymer Cored

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