Biocompatible, Free-Standing Film Composed of Bacterial Cellulose

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Biocompatible, Free-Standing Film Composed of Bacterial Cellulose Nanofibers/Graphene Composite Lin Jin, Zhiping Zeng, Shreyas Kuddannaya, Dingcai Wu, Yilei Zhang , and Zhenling Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11241 • Publication Date (Web): 15 Dec 2015 Downloaded from http://pubs.acs.org on December 18, 2015

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ACS Applied Materials & Interfaces

Biocompatible, Free-Standing Film Composed of Bacterial Cellulose Nanofibers/Graphene Composite Lin Jin,†,‡,§ Zhiping Zeng, § Shreyas Kuddannaya, § Dingcai Wu, ‡* Yilei Zhang, §* Zhenling Wang†* †

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

University, Zhoukou 466001, P. R. China ‡

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

Engineering, Sun Yat-sen University, Guangzhou 510275, P. R. China. §

School of Mechanical & Aerospace Engineering, Nanyang Technological University, 50

Nanyang Avenue, Singapore 639798, Singapore

ABSTRACT: In recent years, the graphene films have been series of wide applications in biomedical area due to several advantageous characteristics. Currently, these films are derived from graphene oxide by chemical or physical reduction methods, which results in a significant decrease in surface hydrophilicity although the electrical property could be greatly improved due to the reduction process. Hence, the comprehensive performance of the graphene films showed practical limitations in the biomedical field due to incompatibility of highly hydrphobic surfaces to support cell adhesion and growth. In this work, we present a novel fabrication of bacterial cellulose nanofibers/reduced graphene oxide film (BC-RGO film) by bacterial reduction method. BC-RGO films thus prepared, maintained excellent hydrophilicity coupled while electrical properties were improved by bacterial reduction of graphene oxide films in culture. The human marrow mesenchymal stem cells (hMSCs) cultured on these surfaces showed improved cellular

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response with higher cell proliferation on the BC-RGO film compared to free-standing RGO film without the nanoscale fibrous structure. Further, the cellular adhesion and proliferation were even comparable to that on the tissue culture plate, indicating that the BC nanofibers play a critically contructive role in supporting cellular activities. The novel fabrication method greatly enhanced the biochemical activity of the cells on the surface which could aid in realizing several potential applications of graphene film in biomedical area, such as tissue engineering, bacterial devices, etc. KEYWORDS: graphene, bacterial reduction, cellulose nanofibers, hMSCs, tissue engineering


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1. INTRODUCTION Graphene, a two-dimensional carbon monolayer material, has sparked tremendous research interest in the energy, environment and biomedical applications because of its extraordinary electrical, chemical, optical and mechanical properties.1-14 These properties have been tapped in a wide array of applications in fields such as supercapacitors,15, 16 lithium ion batteries,17, 18 tissue engineering studies,19,

20

bacterial electronic devices,11,

21

bacterial sensors,22,

23

and drug

delivery,24 etc. In recent years, various approaches including filtration, self-assembly, chemical vapor deposition and others have been employed to fabricate graphene based nanofilms with desired physicochemical or biochemical properties to cater for specific practical applications.25-29 These methods have notably aimed at improving the development of mutifunctional substrates for application in the area of bio-medicine applications such as diagnostic bio-sensors30 and mammalian cell based investigations applicable in tissue engineering and regenerative medicine.31 However, one of the key issue and a limiting factor concerning the applicability of graphene based films in these areas is the substantial decrease in surface hydrophilicity which is accompanied with the usual improvements in electrical property induced during the reduction process.32 Thus, the biocompatibility and biological interactions of the graphene film can not meet the wide range of potential applications in biomedical field. Recently, surface functionalization of graphene films through chemical modification and reduction methods have been considered and tuned to achieve specific biofuntionalization on graphene surface.33-37 Although these fabrication methods significantly improved the bio-activity of the graphene film, through the immobilized biomolecular interfaces, which could aid potential applications,38-40 current methods are still not fully defined and understood to satify the biomedical applications concerning cell growth and development on a graphene based



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bio-compatible interface. Hence, novel preparation processes of graphene film need to be explored to satisfy the complex requirments of the organisms concerning tissue and cellular growth, maturation, differentiation and regeneration. In this study, we report a facile yet efficient bacterial reduction method using Gluconacetobacter intermedius BC 41 (G. intermedius BC-41) to develop a class of novel graphene film for biomedical applications. The bacterial reduction process resulted in an improved, well maintained surface hydrophilicity along with the enhancement of electrical properties. Thus several drawbacks of conventional physical and chemical approaches could be effectively overcomed. Furthmore, this method is convenient to scale up, eco-friendly, and capable of constructing free-standing BC-RGO film with excellent electrical property, promising hydrophilicity and desired biocompatibility. More importantly, compared with a conventional RGO film, the as-prepared BC-RGO film significantly improved the cellular activities on the surface with enhanced cell adhesion and proliferation. Considering the highly enriched integration of the unique physical and chemical properties, we hope that the prepared BC-RGO film could propel the advancement in biotechnological areas, concerning the development of electrically active substrates for bio-medical device fabrication, tissue engineering, biosensors, etc. 2. EXPERIMENTAL SECTION 2.1. Materials Na2HPO4·12H2O and NaOH were purchased from Guangzhou Chemical Co. Deionized water (18 mΩ) from the milli-Q setup was used for performing all the electrochemical measurements. 2.2. Fabrication of BC-RGO Films


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GO was prepared by following previous reported methods.41,42 Deionized (DI) water was used in the experiments, and GO sheets dispersion (1mg/mL) was achieved through the GO aqueous solution by ultrasonication method (using the ultrasonic cleaner at a frequency of 40 kHz and a output power of 100W). The prepared dispersion was centrifuged at a speed of 3000 rpm for 0.5 h to remove any possible traces of unexfoliated graphene oxide. G. intermedius BC-41 was cultured according to previous reported method.43 Briefly, G. intermedius BC-41 was routinely grown in Schenk and Hildebrandt (SH) medium (glucose 20 g/L, peptone 5 g/L, yeast 5 g/L, Na2HPO4·12H2O 2.7 g/L, citric acid 1.5 g/L), with pH 5.0 at 28 °C, in a shaking incubator with 120 rotations per minute (rpm). The aliquots of 10% SH medium culture with G. intermedius BC-41 were transferred into 100 mL mixture of SH medium (90%) and graphene oxide solution (10%) in 500 mL flasks and then cultured in a static incubator at 28 °C for 14 days. Finally, the film was washed by DI water and soaked in 10 wt% NaOH solution at 100°C for 2 h, and then frozen at -20°C for 24 h and dried by freezing dryer. 2.3. Structural and Chemical Characterization. The morphology and thickness of the prepared GO sheets were measured by a tapping-mode CypherTM atomic force microscope (AFM) and operated at the ambient conditions. The morphology of BC-RGO film was characterized by scanning electron microscope (SEM, Hitachi S-4800) at an acceleration voltage of 10 kV after the samples mounted on the conductive adhesive and sputter-coated by gold and palladium. The mechanical properties of BC-RGO film were evaluated using a micro-tensile testing system (Sans-GB T528, Shen Zhen, China) at a speed of 1mm/min according to our reported method.16 The chemical compositions were examined by FTIR with a ATR model (Nexus, Thermo, Scientific, USA), Raman spectrum



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(RIGAKU Co., Japan) obtained under a green laser (532 nm) with a power of 2 mW, and XRD pattern (Bruker D8 Advance, Germany) with Cu-Kα radiation (λ = 0.15406 nm). 2. 4. Biocompatibility Analysis hMSCs were obtained

from side population of human PLC/PRF/5 cell line (ATCC,

Manassas, VA, USA) with flow cytometry of Hoechst 33342 (Invitrogen, Carlsbad, CA, USA). The obtained hMSCs were kept in complete dulbecco's modified eagle medium (DMEM), and used for the following experiments within 2 h. The BC-RGO film was cut into 16 mm discs and placed into 24-well plates,19,20 and then the prepared samples were sterilized using a mixture of 30% phosphate buffered saline (PBS) and 70% ethanol solution for 6 h, followed by washing 3-5 times using PBS solution. hMSCs were cultured on the substrates according to our previous work.44 Briefly, hMSCs were cultured on the BC-RGO film, RGO film and TCPs at a cell density of 1.6 × 104 cells/well with 0.5 mL DMEM supplemented with 10% (v/v) Fetal Bovine Serum (FBS). The Cell-substrates were incubated under a humidified incubation condition of 37ºC and 5% CO2. Cell viability on the BC-RGO film was evaluated by fluorescence images. Hoechst 33258 (5µg/mL, Sigma-Aldrich) and Alexa Fluor 546®phalloidin (5µg/mL, Sigma-Aldrich) were used to visualize cellular nuclei and cytoplasm, respectively. All cell-substrates were fixed by 3.7% paraformaldehyde for 30 min prior to staining and the fluorescence images were characterized by a Leica TCS-SP2 Confocal Microscope (Leica, Germany) and analyzed using TCS Leica Software 2.61. Cellular morphology on the BC-RGO film, RGO film and TCPs was evaluated by SEM images. The cell-substrates were fixed by 3% glutaraldehyde for 12 h and dehydrated by ethanol/water solution with various concentrations for 30 min,16 following which the


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cell-substrates were dried in air. The samples were sputter-coated by gold and palladium with a thickness of 10 nm and SEM images were taken by Hitachi S-4800. Cellular adhesion and proliferation were quantified at the specified time points by a DNA analysis method.45, 46 Briefly, at each time point, PicoGreen® DNA quantification (Quant-It Picogreen, P7589, Invitrogen) reagent was incubated with each lysate for 5 min to assess the DNA content of the samples according to the manufacturer’s instructions. Fluorescence of the substrates was evaluated at 485/535 nm by a Victor3 multilabel fluorescence plate reader (PerkinElmer, USA). The cell number was calculated from the absorbance standard of the known cell concentration. 2.5. Statistical Analysis. All samples were characterized in quadruplicate at least three independent times, and the results expressed are representative of these data sets. All data were expressed as mean ± standard deviation. Statistical analyses were performed with the t-test. All values were analyzed for statistical significance using the SPSS software (version 11.0). 3. RESULTS AND DISCUSSION The prepared GO sheets in the aqueous solution exhibit irregular shapes and lateral dimensions range from ~100 nm to ~2 µm (Figure S1). The average thickness of the obtained GO sheets is about 1nm, slightly thicker compared to the monolayer graphene sheet, which maybe attributed to the oxygen functional groups bearing on the basal planes and edges of the hydrophilic GO sheets.47,48 G. intermedius BC-41 has a high ability to produce bacterial cellulose nanofibers.41 Moreover, unique nano-morphology of these obtained cellulose nanofibers make them have high



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water-holding capacity, great elasticity and high wet strength. These integrated performance enable them to be used successfully in applications within the biomedical field.49 In addition, previous reports indicated that GO can act as a terminal electron acceptor for environmental bacterial.37 Thus, in this study, we used G. intermedius BC-41 to convert graphene oxide and form reduced graphene film. The fabrication procedure of BC-RGO film (Figure 1) includes three steps: G. intermedius BC-41 culture, self-assemble (incubation and biochemical reduction) and freeze drying. For bacteria culture, G. intermedius BC-41 was selected based on the application requirements. As a demonstration, a mixed solution of GO solution, G. intermedius BC-41 and medium was prepared. Then, this

solution was stored in static incubator at 28°C for 14 days culture

conditions, leading to the in-situ self-assembly and subsequent reduction of GO sheets onto the surface of the bacterial cellulose nanofibers to form a smooth and condensed RGO film. After taking out from the culture solution and washing by DI water, BC-RGO film was soaked and washed using NaOH solution (10 wt %) at 100ºC for 2 h. Firstly, we investigated the G. intermedius BC-41 without GO solution for the formation of the bacterial cellulose nanofibers. After 2 weeks in culture, the SEM image shown in the Figure 2A, clearly indicated that the G. intermedius BC-41 could enable the formation of bacterial cellulose nanofibers. With GO added into the culture medium, and subsequntly followed by the self-assembly and drying processes, the morphology of the BC-RGO film presents a nanoscale pattern due to the interconnection each other of reduced graphene oxide sheets (Figure 2B). The RGO sheets were tethered together by the bacterial cellulose nanofibers and forming the RGO film. At the edge of the hence formed RGO films the interconnected network formed by the bacterial cellulose nanofibers to form a RGO film structure, could be clearly observed (Figure S2). Furthermore, the


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SEM images of the positive face (Figure S3) also clearly demonstrated the cross section surface structure of the RGO film. This cross sectional view,

indicates that the RGO sheets were

connected each other by G. intermedius BC-41 and/or RGO itself and then formed the film through the layer by layer stacking of these interconnected networks. These results confirmed that the BC-RGO film could be effectively prepared by the formation of bacteria mediated cellulose nanofibers connection and the biochemical reduction process. Raman spectroscopy, XRD pattern, and FTIR spectrum were used to characterize the chemical compositions of the BC-RGO film. The XRD patterns of BC-RGO film indicate that a strong reflection with peak at 2θ = 24.78° appears, which could be attributed to the (002) reflection of RGO (Figure 2C). Notably, the characteristic peak shifted from reflection peak of GO (9.9°) to 24.78°, which is likely due to the elimination of the oxygen containing groups in the lower d-spacing.50-52 FTIR spectrum (Figure 2D) confirmed that the dominant oxygen-containing groups, including -OH and C=O stretching of carboxylic acid and ester groups, have almost been removed after the bacterial reduction.53-55 The Raman spectrum was shown in Figure 3, which indicated that after the bacterial reduction of GO sheets, the D peak obviously increases and the G band of BC-RGO film is demonstrated at 1595 cm-1. These Raman peaks of BC-RGO film are similar to that in the pristine graphite,47 confirming the successful reduction of BC-RGO film using the G. intermedius BC-41. These results combining with the SEM images (Figure 2B) indicate that the BC-RGO film has been successfully fabricated by the union of self-assembly and bacterial reduction. This reduction could be attributed to the metabolism of G. intermedius BC-41 during which the extracellular electrons were transfer to GO, which shows the ability of the bacterial culture to reduce GO, thereby inducing an efficient biodegradation of GO under ambient conditions.



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The mechanical properties of the BC-RGO film were characterized under tensile loading at room temperature (Figure 4A) with typical fracture strengths of 1.32±0.1 MPa, at ultimate elongations of 4.23±0.16%, respectively. The tensile stress was slightly lower compared to the RGO film converted by chemical method (fracture strengths: 13.6±0.5 MPa, ultimate elongations: 3.8±0.15%);19 but the unique “interconnected” structure with aligned graphene building blocks and the stacking of GO sheets make BC-RGO film still have good mechanical performance, which is enough to ensure that the BC-RGO film could function under the harsh mechanical environments within living organisms or flowing liquid. These mechanical features further ensure its applicability as electroactive substrates/constructs for tissue engineering, bacterial sensors, drug delivery, and implanted electrodes. The electrochemical performance of BC-RGO film was evaluated in the PBS solution (0.1 M, pH=7.4) by cyclic voltammetry (CV) using a three-electrode system. BC-RGO film was used as a working electrode, and a platinum wire was used as a counter electrode against an Ag/AgCl reference electrode. BC-RGO film (2 cm × 2 cm) was immersed in PBS solution, and the voltammograms were recorded with a scan rate of 50 mV/s. Although the electrical-chemical property could be seen to be not as good as RGO films obtained from chemical method, the CV curve of bacteria RGO film (Figure 4B) still exhibits a high charge carrying capacity for a given voltage. This excellent electrical property of BC-RGO film maybe attributed to two factors: the large surface area and the large number of π-π conjugated bonds, which generates a near perfect spatial continuity over the entire surface of the RGO layer, permitting efficient electron transfer. Thus, the as-prepared BC-RGO film could provide an appropriate range of current for electrical stimulation atmosphere in cell culture or can collect the physiological signals, functioning as a bacterial sensor.


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For cell culture substrates, a good hydrophilic property is required to facilitate efficient cell adhesion on surfaces mediated by the cell cytoskeleton adhesion machinery.56 Figure 4C shows the water contact angles of BC-RGO film and RGO film. The results demonstrate that the BC-RGO film has good hydrophilicity (14.3±1.3°) in comparison with the high hydrophobicity of the RGO films (92.94±3.2°), which could be most likely attributed to the high density of bacterial cellulose nanofibers embedded in the BC-RGO film. These properties make BC-RGO film show great application prospect in biomedical field especially in understanding and mediating cell based phenomenon like hMSC cell proliferation and differentiation which are dependent on efficent cell anchorage on substrate surfaces.57 For celluar response of BC-RGO film, hMSCs were used to evaluate the adhesion and proliferation of cells on the BC-RGO film. Figure 5 shows the normalized measurements of cells on the BC-RGO film, RGO film and TCPs. After 6 h culture, the cellular attachment of hMSCs on the TCPs, the BC-RGO film, RGO film were found to be 94.06%, 94.37% and 71.9%, respectively, which indicate that the hydrophilic BC-RGO films could positively influence cell adhesion compared to the hydrophobic RGO film. After 1 day culture, the cellular number of hMSCs on the BC-RGO film, RGO film and TCPs are 2.56×104, 1.89×104 and 2.51×104, respectively. Which are significantly higher than that on the RGO films. On day 3, the increase in cell count on the BC-RGO films is still much higher than that on the RGO film and even higher than on the TCPs, which demonstrates that hMSCs on the BC-RGO film remained metabolically active compared to RGO film and posessed vigourous proliferation rate. After 7 days in culture, the proliferation of hMSCs on the BC-RGO film showed a notable increase. The cellular number on the BC-RGO films increased to 9.65×104, still higher than that on the RGO film and TCPs (only 6.891×104 on the RGO films and 9.15×104 on the TCPs). These results



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strongly suggested that the BC-RGO film had a significant effect on the cell adhesion and proliferation, and could provide a preferable cell growth microenvironment, thereby showing a great potential in applications for tissue engineering and biomedical devices in which particularly high electrical conductivity and/or biocompability are required. hMSCs were cultured 3 days on the BC-RGO film with a cell density of 1.6×104/well for detecting cellular activity by confocal microscope, and RGO films and TCPs were set as the control groups. F-actin and nuclei of the hMSCs on the various substrates were visualized by staining with Alexa Fluor 546® phalloidin and Hoechst 33258, respectively. hMSCs cultured on the BC-RGO film displayed much better cell attachment and cell retention (Figure. 6) compared to that on RGO films and even better than that on the TCPs. In comparison, hMSCs on the TCPs spread freely due to its isotropic surface allowing hMSCs to grow in all directions, while hMSCs on the RGO films showed a shrinked morphology and aggregation due to its poor biocompatibility. These results indicate that the BC-RGO film has positive effect on cell spreading and activity, and bacterial education of the GO films during fabrication process assisted with layer by layer tethering of bacterial cellulose nanofibers could provide better survival micro-environment for cell growth compared to the chemical reducation. A detailed morphology of hMSCs cultured on BC-RGO film, RGO films and TCPs was evaluated by SEM after 3 days in culture. The hMSCs on the TCPs displayed a large number of cellular extentions which were spread across randomly on the isotropic surface of TCPs (Figure. 7A). The morphology of hMSCs on BC-RGO film (Figure. 7C) showed that the cells adhered tightly on the surface of bacterial functionalized film and formed integrated cell-film constructs, so that it was difficult to visually distinguish the cellular morphology between single hMSCs and the surface of BC-RGO film. Furtherfore, the cells on these constructs exhibited wide cell-cell


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and cell-film contact, which provide positive help for the maintenance of cell activity and growth. In contrast, hMSCs cultured on RGO film appeared loosely adhered on the surface with an unclear, clumped morphology (Figure. 7B). This reveals that BC nanofibers of the BC-RGO films enhanced hMSCs adhesion and speading, which also implied a positive interaction among hMSCs and with the surface of the BC-RGO film. 4. CONCLUSION In conclusion, we successfully prepared a novel class of high-performance BC-RGO film by bacterial reduction using G. intermedius BC-41. The obtained BC-RGO film exhibits valuable nano-fibrous structure, excellent mechanical, electrical and hydrophilic properties. In vitro experiments indicate that BC-RGO film could effectively enhance cells adhesion, growth and proliferation of hMSCs. Moreover, BC-RGO film was shown to be highly supportive for the interaction between the cells and the surface of the film, and has a much better cellular response when compared to the RGO film. We believe that the BC-RGO films prepared through the above discussed novel technique might greatly aid several potential applications as substrates for engineered bacterial scaffolds with electrical stimulation, devices and biosensors for transmitting or collecting physiological signals. AUTHOR INFORMATION Dr. L. Jin, Prof. Z. L. Wang* †

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 Prof. D. C. Wu*



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‡

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Materials Science Institute, School of Chemical and Chemical Engineering, Sun Yat-sen

University, Guangzhou, 510275, P. R. China E-mail:[email protected]. Z. P. Zeng, S. Kuddannaya, Prof. Y. L. Zhang* §

School of Mechanical & Aerospace Engineering, Nanyang Technological University, 50

Nanyang Avenue, Singapore 639798, Singapore 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. 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). Y. Zhang ackonwledes Tier-1 Academic Research Funds from the Singapore Ministry of Education (RGT 30/13, RGC 6/13 and RGC 1/14). REFERENCES (1) Zhao, J. P.; Pei, S. F.; Ren, W. C.; Gao, L. B.; Chen, H. M. Efficient Preparation of Large-Area Graphene Oxide Sheets for Transparent Conductive Films. ACS Nano 2010, 4, 5245-5252.


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Figure 1. Schematic diagram depicting the fabrication and applicaion of BC-RGO film G, which includs: (1) intermedius BC-41 culture, (2) self-assemble (incubation and biochemical reduction) and (3) freeze drying. Bacterial cellulose nanofibers generated using G. intermedius BC-41 were embedded in BC-RGO film and seeded with human marrow mesenchymal stem cells (hMSCs). hMSCs cultured on the BC-RGO film show enhanced cell adhesion and proliferation.



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Figure 2. SEM images of (A) the bacterial cellulose nanofibers, (B) BC-RGO film. (C) XRD patterns and (D) FTIR spectra of GO film, BC nanofibers and BC-RGO film.


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Figure 3. The Raman spectrum GO and BC-RGO films.



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Figure 4. (A) Mechanical tensile strain-stress, (B) CV curves and (C) water contact angles of BC-RGO film and RGO film. The mechanical data of RGO film was from ref. 19.


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Figure 5. The adhesion and proliferation of hMSCs on the BC-RGO film, RGO film and TCPs, respectively.



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Figure 6. Fluorescence images of hMSCs cultured on TCPs, RGO film and BC-RGO film.


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Figure 7. SEM images of hMSCs on the various substrates: (A) TCPs, (B) RGO film and (C) BC-RGO film.



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Table of Contents Graphic


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Biocompatible, Free-Standing Film Composed of Bacterial Cellulose

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