Synergistic Effects of Conductive Three-Dimensional Nanofibrous

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Synergistic Effects of Conductive 3D Nano-fibrous Micro-environments and Electrical Stimulation on the Viability and Proliferation of Mesenchymal Stem Cells Lin Jin, Qinwei Xu, Shuyi Wu, Shreyas Kuddannaya, Cheng Li, Jingbin Huang, Yilei Zhang, and Zhenling Wang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00455 • Publication Date (Web): 09 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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ACS Biomaterials Science & Engineering

Synergistic

Effects

of

Conductive

3D

Nanofibrous

Micro-environments and Electrical Stimulation on the Viability and Proliferation of Mesenchymal Stem Cells Lin Jin,†, ‡ Qinwei Xu,‡ Shuyi Wu,⊥ Shreyas Kuddannaya, ‡ Cheng Li, ‡ Jingbin Huang, † 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 ⊥

Department of Prosthodontics, Guanghua School of Stomatology, Hospital of Stomatology,

Sun Yat-sen University, Guangzhou, 510055, P. R. China

*Corresponding author. E-mail: [email protected], [email protected], Tel.: +86-394-8178518; Fax: +86-394-8178518.

ACS Paragon Plus Environment


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ABSTRACT: In recent years, three-dimensional (3D) scaffolds have proven to be highly

advantageous in mammalian cell culture and tissue engineering compared to 2D substrates. Herein, we demonstrated the fabrication of a novel 3D core-shell nanofibers (3D-CSNFs) using an improved electrospinning process combined with in situ surface polymerization. The obtained 3D nanofibrous scaffold displayed excellent mechanical and electrical properties. Moreover, the cotton-like 3D structure with large internal connected pores (20-100 µm), enabled cells to easily infiltrate into the interior of the 3D scaffold with a good spatial distribution to mimic the ECM-like cell microenvironments. Stable cell-fiber composite constructs were formed in the 3D-CSNFs with relatively higher adhesion and viability compared to 2D-CSNFs. Furthermore, the human mesenchymal stem cells (hMSCs) cultured on conductive polymer coated electrically active 3D nanofibers responded with a healthy morphology and anchorage on the fibers with relatively higher viability and proliferation under electrical stimulation (ES). This study demonstrates the successful fabrication of 3D-CSNFs and the constructive interaction of the 3D-microenvironment and subsequent electrical stimulations on hMSCs, thereby holding a promising potential in tissue engineering and regenerative therapies aided by electro-stimulation based differentiation strategies. KEYWORDS: electrospun nanofibers, 3D scaffold, electrical stimulation, hMSCs, tissue engineering 1. INTRODUCTION Functionalized nanofibers have spurred a tremendous research interest in recent years.1-10 Recently, various nanofibers with outstanding performance have been prepared for applications in biomedicine,11-13 energy storage,14-16 electronic devices,17 sensors,18 and others.19-24 In these applications, functionalization of native nanofibers could be effectively


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tuned to achieve the desired surface interactions and reactivity or to enhance the performance of the native nanofibers. In this regard, designing flexible nanofibers with conductive shell has obtained particular attention in tissue engineering. Recently, exogenous electrical fields have been shown to regulate calcium influx25 and ROS generation26 in stem cells and direct the stem cell fate towards cardiac27 and neuronal lineages28-30. Efforts have been made to synthesize electrically active material interfaces which can closely interact with the stem cells while uncompromisingly considering the topographical and biochemical complexities of stem cell microenvironments. With this motivation, electrically active nanofibers

possessing

advantageous

mechanical

and

topographic

properties

and

biocompatibility have been fabricated with an ultimate aim to effectively translate model studies on well-defined topographical features into scaffold materials that could be used for stem cell aided nerve and cardiac tissue regeneration. Conductive polymers such as polypyrrole (PPy) are known to provide a biocompatible interface favorable for both cell survival31, 32 and electrical modulation of cell behavior.33-35 PPy coating on nanofibers could mimic natural extracellular matrix (ECM) to provide a favorable microenvironment and govern cellular responses, including cell adhesion, migration, proliferation, and differentiation of a wide range of cell types. Moreover, the conductive coating could still retain the mechanical properties (E.g, elasticity, pore size distribution and micro and nanoscale fibrous architecture) of the native fibers.36-41 In recent years the simplicity, versatility, and ease of scale-up of the electrospinning technique have been widely exploited for diverse purposes. The morphology and structure of electrospun nanofibers can be easily controlled by simply altering the electrospinning conditions such as polymer concentration, feed rate, collector distance, and applied



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voltage.42-47 Previous reports have shown that two-dimensional (2D) conductive nanofibers could be prepared by electrospinning as a conductive cell culture substrate to enable electrical stimulation.48-50 However, such 2D fibrous mesh systems do not adequately mimic the in vivo 3D extracellular microenvironments to support cellular growth and proliferation. Particularly, the densely packed 2D nanofibers impeded the cell penetration into the interior accesses of the scaffolds. To overcome these limitations, a great deal of focus has been paid to develop novel nanofiber fabrication processes. In this study, we have developed a facile and convenient method to fabricate conductive 3D core-shell nanofibers using an improved electrospinning process combined with direct surface polymerization of conductive PPy. The developed method is easy to scale up and capable of constructing free-standing 3D-CSNFs with excellent electrical and mechanical properties. Most importantly, when compared with the 2D conductive nanofibers, the as-prepared 3D-CSNFs showed enhanced biocompatibility with a significant improvement in penetration, adhesion, and proliferation of the human mesenchymal stem cells (hMSCs). Moreover, the 3D-CSNFs could be an excellent in-vitro platform to study and facilitate electro-stimulation based enhancement and direction cellular growth, metabolism, over physiological regulation and eventual cell lineage modulation. Considering the unusual integration of the nanofibrous structure and the outstanding electrical, mechanical and biocompatible properties, we believe that the obtained 3D-CSNFs could have a broad range of applications in tissue engineering such as electrical modulation of cells and tissue functions, non-invasive control of cell fate and in devising sustained and controllable drug release based therapies. 2. EXPERIMENTAL SECTION


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2.1 Materials Polyacrylonitrile (PAN, MW = 100k) was provided by Daigang Polymer (Jinan, China), N, N-dimethyl-formamide (A. R., Guangzhou Chemical Reagent Co.). Pyrrole and ferric chloride were purchased from Sigma-Aldrich (Singapore). The DMEM and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (USA). 2.2 Fabrication of 3D-CSNFs The fabrication of 3D core-shell conductive nanofibers includes three steps: (I) Electrospinning nanofibers into DI water solution with FeCl3 and Pyrrole, (II) The pyrrole was polymerized under ultrasonic treatment, (III) 3D-CSNFs prepared by freeze drying (Figure 1). Firstly, the PAN precursor solution was obtained by dissolving (Mn=100k) in N, N-dimethyl-formamide (DMF) with 10 wt%, under constant stirring until the mixture was rendered clear, viscous and homogenous. Subsequently, the mixed solution was fed into a syringe capped with 0.22 gauge blunt-tipped needle and driven by a syringe pump (Langer CO., Baoding, China) at a controllable feed rate of 1.0 mL/hour. The distance between the tip of the syringe needle and the collector was fixed at 10 cm with a voltage of 15 kV (Dongwen High Voltage, Tianjing, China). The produced nanofibers were collected in a beaker (1L) containing 400 mL pyrrole aqueous solution (0.02 M). Eventually, 400mL FeCl3 aqueous solution (0.168M) was added to the aqueous collection solution and the mixture was ultrasonicated (50 KHz, 400W) for 1 h. Finally, the PPy coated nanofibers were soaked and thoroughly rinsed in the DI water to remove the residual chemicals and stored at -20ºC in sterile 50 ml tubes for 24h before freeze-drying. 2.3 Characterization of 3D-CSNFs



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The SEM of 3D-CSNFs were examined by a Hitachi S4800 field-emission SEM system at an accelerating voltage of 15kV; transmission electron microscope (TEM) was performed on JEOL-2100F with a field emission gun operating at 200 kV. The chemical compositions were examined by Raman spectrum(Nexus, Thermo, Scientific, USA) and XRD pattern (Bruker D8 Advance, Germany) with Cu-Kα radiation (λ = 0.15406 nm). The Fluorescence images were obtained using a Confocal Microscope (Leica TCS-SP2, Leica, Germany) with TCS Leica Software 2.61. 2.4 Cell culture and characterization hMSCs were obtained and cultured to evaluate cellular response on the nanofibers. The hMSC culture was performed according to our previously reported protocol.9 Briefly, 3D-CSNFs were cut and placed in 24-well tissue culture plate chambers, and sterilized using phosphate buffered saline (1× PBS) and ethanol aqueous solution (70% ethanol and 30% PBS) for 6 hours, followed by washing several times with 1× PBS solution. hMSCs were seeded onto or into the various substrates at a cell density of 1.2× 104 cells/well with 0.5 mL Dulbecco’s modified eagle medium (DMEM) medium supplemented with 10% (v/v) FBS. All the cell constructs were incubated under humidified condition with 5% CO2 at 37°C. To assess cell viability and morphology in the 3D-CSNFs after five days’ culture, samples were incubated with 5µg /mL Phalloidin-FITC (green, Sigma-Aldrich) and 5 µg /mL Hoechst 33258 (blue, Sigma-Aldrich) in culture medium for 20 min at 37°C, and then fixed using 3.7% paraformaldehyde for 30 min and imaged by confocal fluorescent microscopy.


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Detailed cellular morphology of the 3D-CSNFs was characterized using SEM. After five days’ culture, the cells were fixed by 3% glutaraldehyde for 12 h and dehydrated using serially increasing ratio of ethanol/water (v/v) concentrations according to our previously reported methods.37,38 The samples were then dried in air overnight. Platinum/palladium was coated on the various samples with 10 nm in thickness using a sputter coater before SEM imaging. Cellular adhesion and proliferation were quantified at specialized time points based on a DNA analysis method.51,52 PicoGreen® DNA quantification (Quant-iT Picogreen, P7589, Invitrogen) was used to measure the DNA content of the various samples incubated for cellular proliferation. After 6h, 1, 3, and 7 days incubation, hMSCs were lysed using 1% triton X-100 and subjected to several freeze-thaw cycles. DNA content was calculated from the lysates (25 µL) using PicoGreen® DNA according to the manufacturer’s instructions. Briefly, 75 µL of PicoGreen® reagent was incubated with each lysate protected from light for 5 minutes at room temperature. Fluorescence of the samples was measured at 485/535 nm using a Victor3 multilabel fluorescence plate reader (PerkinElmer, USA). The DNA content of samples was assayed and determined with reference to a standard curve. The cell numbers were determined from corresponding DNA concentration values and plotted. To evaluate the effect of conductive nanofibers surface on hMSCs, cellular collagen content was quantified on day 7 according to the previously reported method.46,47 Briefly, the cell-laden nanofiber constructs were washed using sterile PBS, and then incubated using papain digestion buffer (125 µg/ml Papain, 5mM L-cysteine, 5mM EDTA and 100mM Ha2HPO4, PH=7.5) at 70°C for 16 h. After digestion, the solution was mixed with HCl (12M) for hydrolysis at 120°C for 3 h. The hydrolyzed solution was then transferred to



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a 96-well plate in an oven for drying. Collagen content was estimated by hydroxyproline measurement (Hydroxyproline Assay Kit, Sigma, Singapore) as per the manufacturer’s protocol. Deionized (DI) water was used as a blank in the assay measurements. 2.5 Cell culture under electrical stimulation Electrical stimulation of hMSCs was performed in the 3D-CNFs with a self-made bioreactor. Cells were seeded into 3D-CNFs after 24 h, the cell chambers were closed with a sterilized lid assembled with stainless steel electrodes so that the scaffolds could come in contact with the electrodes. A 100 Hz pulsed electrical potential of 100 mV/cm was applied across two electrodes for 4 h in the incubator by an AFG3022C function generator (Tektronix, USA) every day for one week. Cells were analyzed every 24 h following electrical stimulation cycles. All data were noted as mean ± standard deviation, and statistical analyses were performed with the standard student’s t-test. All the obtained quantitative data were analyzed by the SPSS statistical software (version 11.0). 3. Results and Discussion 3.1 Fabrication and characterization of 3D-CSNFs 2D-CSNFs could be easily obtained (Figure 2A) using conventional electrospinning process. The nanofibers in fibrous mat appeared to be closely packed together before and after coating PPy layer. However, the 3D nanofibers obtained using improved electrospinning process and freeze-drying method demonstrated a fluffy and porous morphology. After coating PPy layer using in situ surface polymerization, the native 3D structure could be still retained (Figure 2B). These results indicate that the 3D-CSNFs could be successfully


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fabricated

using

our

improved

electrospinning

process

combined

with

surface

polymerization. The obtained 3D-CSNFs have a loosely interwoven internal structure with pore size ranging between 20-100 µm (Figure 3B). Compared to closely packed nanofibers in conventional 2D-CSNFs (Figure 3A), the majority of the nanofibers in the 3D-CSNFs were distributed in the 3D space with few interconnected nanofibers, i.e., forming a deeper and interconnected network along a 3D space. The SEM images obviously indicated that the 2D-CSNFs could only provide the upper surface to promote cell growth, while the loosely packed nanofibers in 3D-CSNFs could enable cells to easily penetrate into the deep interior of the 3D-CSNFs, thereby grow in a natural ECM like 3D cellular microenvironment. Moreover, a large number of interconnected micro-pores of the 3D-CSNFs allow the transport of nutrients (from the medium) and the metabolic wastes (from the cells) to flow into and out of the scaffold, respectively, favoring the long-term viability of the adhered cells. The microstructure details of individual nanofibers in the 3D-CSNFs were revealed in the TEM and high magnification SEM images (Figure 3C, D). The results indicated that the desired core-shell structure could be successfully fabricated and the PPy outer layer coated on the PAN nanofiber could be clearly observed. The Raman spectra (Figure 4A) of 3D-CSNFs shows that the characteristic band at 1330 cm-1 for the presence of C-N stretching, and the band at 1550 cm-1 for the C=C stretching, which implied that some pyrroles in the PPy chains could not be completely doped. In addition, the XRD pattern of 3D-CSNFs indicates that PPy layer on the surface of nanofibers was amorphous solid because of the broad peak around 2θ = 21.3°, and the peak at 26.2° which appears to match well with the standard XRD data (Figure 4B) which were



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consistent with the previous reports.53,54 The prepared 3D-CSNFs demonstrated excellent electrical and mechanical properties. The electrochemical performance of the 3D-CSNFs was characterized using the cyclic voltammetry (CV) in the 0.5M Na2SO4 solution with a three-electrode system, i.e., the 3D-CSNFs as the working electrode, a platinum wire as a counter electrode and Ag/AgCl as the reference electrode. The 3D-CSNFs (20.6 mg) were immersed in the Na2SO4 solution. And the voltammograms were recorded with a scan rate of 50mV/s. The CV spectrum (Figure 4C) indicated that the 3D-CSNFs exhibited high charge carrying capacity for a given voltage and excellent electrochemical property. It is most likely attributed to the large surface of 3D-CSNFs, which promotes both the incorporation of dopant ions into the matrix and formation of packed PPy chains, thus permitting efficient electron transfer between the polymer chains. The mechanical properties of the 3D-CSNFs were characterized using tensile compression tests (Figure 4D), which showed a compression stress around 1.1 kPa under 40% strain, i.e., a Young’s modulus around 2.75 kPa. Moreover, the 3D-CSNFs could recover its original state after releasing the stress, which may be attributed to synergistic effects of the core-shell structure and the compact stacking of PPy layer on the PAN nanofibers. 3.2 Biocompatibility analysis To explore the cytocompatibility of the nanofibers and to assess the cell morphology and growth in nanofibrous environments, hMSCs with a density of 1.2×104/well were seeded onto the sterile 3D-CSNF scaffolds. In order to evaluate the cellular response to different spatial micro-architectures within the nanofibers, hMSCs were cultured on/in the 2D-CSNFs and 3D-CSNFs for 5 days. The cytoskeletal F-actin and nuclei staining (Figure 5) indicated


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that the hMSCs could deeply penetrate into the scaffold and uniformly distribute in the interior of the 3D-CSNFs. In addition, the cells cultured in the interior of 3D-CSNFs displayed much better cell attachment with indications of healthy cell adhesion i.e. higher cell spread area and retention compared to those cultured on the surface of 2D-CSNFs. Furthermore, hMSCs within the 3D-CSNFs showed widely spread network of cells with rich cell-cell contact and well-anchored cell-fiber constructs along the nanofibers, which is essential for subsequent proliferation and cellular activity. However, hMSCs cultured on the 2D-CSNFs displayed a rather round morphology with sparse cell-cell contact and the cells retained at the superficial surface layer. This could be attributed to the densely interconnected nanofibers, which not only provide the nanofibrous cues in all directions at the surface to form the round cell spread but also limit the cell penetration into the interior layers of the 2D-CSNFs scaffold. These results indicate that the porous and fluffy fibrous structure of 3D-CSNFs not only promote hMSCs permeability into the interior of the scaffold with easy access to nutrients for long-term culture but also facilitate cell attachment, alignment, metabolism and proliferation in the 3D microenvironment. A further detailed investigation of the morphology of hMSCs cultured on/in the 2D-CSNFs and the 3D-CSNFs was performed using SEM after 5 days in culture. Figure 6B shows the morphological details and processes of hMSCs in the 3D-CSNFs, which indicated that the cells could adhere to the nanofibers firmly, and grows along the fiber direction, forming well-integrated cell-fiber constructs (Figure 6B). It was difficult to clearly distinguish the cell-nanofiber junctions of single hMSC cells since the cells appeared to wrap tightly around the individual nanofibers. These results demonstrated that the porous and fluffy architecture of the 3D-CNSFs allows easy penetration of hMSCs throughout the



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scaffold. Moreover, cells in these constructs showed clear cell-cell contact regions which could aid in the maintenance of cellular activity and proliferation. In contrast, the hMSCs cultured on the 2D-CSNFs (Figure 6A) indicated that cells displayed rounded and separated cellular shapes, i.e., cells could not closely adhere to surfaces of the nanofibers and the cell-nanofiber construct was loose, which may be attributed to the poor biocompatibility of the 2D-CSNFs surface. These results were in agreement with the results of the fluorescence images, which revealed that the 3D fluffy and porous structure of the 3D-CSNFs is much more helpful for the promotion of cell activity and growth compared to 2D nanofibers. During the 7 days’ culture period, cellular adhesion, proliferation, and collagen content were quantitatively evaluated. After 6h of culture, the cell adhesion ratio of 3D-CSNFs was 90%, whereas that of the 2D-CSNFs was only 83.3%. After 1 day of culture, the cell number of 3D-CSNFs is slightly more than that on 2D-CSNFs. After 3 days of culture, the proliferation of cells in 3D-CSNFs shows a notable increase, i.e., the cell number in the 3D-CSNFs increased by 150% while that of cells on the 2D-CSNFs increased by 115%. On day 7, a striking increase of the cellular amount of the 3D-CSNFs was observed, which is much higher than that on the 2D-CSNFs. Moreover, the collagen value of 3D-CSNFs is also much higher than that on the 2D-CSNFs after 7 days of culture. These results strongly demonstrated that 3D-CSNFs with loosely packed nanofibers can provide a cytocompatible microenvironment to increase hMSCs proliferation and collagen expression. Hence, the obtained 3D-CSNFs could be foreseen as a promising candidate for stem cell-based regenerative therapies and as a platform to further investigate cell responses and cell fate manipulation on bio-active and conductive nanofibers. 3.3 Effect of electrical stimulation on cell morphology and proliferation


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3D-CNFs have excellent electrical property, which can support electrical stimulation as a cell culture scaffold. To evaluate the effects of ES for hMSCs, cells were cultured for 7 days with daily pulsed ES. The fluorescent images of hMSCs with/without ES were shown in Figure 7, which demonstrated that the cells cultured under ES situation displayed much better cellular growth compared to those cultured without ES. Particularly, hMSCs cultured under ES showed rich cell-cell contact and well-developed hMSCs-fibers constructs. Furthermore, the F-action of cells connects together along the scaffold nanofibers to form a dense network. ES can provide physical stimulations for hMSCs and regulate cellular growth behavior in the internal microenvironment of the 3D-CNFs. To evaluate these effects, the proliferation of hMSCs was measured (Figure 8), which indicated that proliferation of cells under ES condition on day 1 was slightly lower compared to cells without ES. After 4 days of culture, the proliferation of cells under ES condition was not significantly different from cells without ES. These results indicated that the daily pulsed ES had no great influence on the proliferation of cells in 3D-CNFs. 4. CONCLUSION In summary, we successfully developed a novel fabrication process for the preparation of 3D polymer cored, conductive-shell nanofibers using a combination of electrospinning and direct in situ surface polymerization. The prepared 3D-CSNFs demonstrate loosely packed nanofibrous architecture, excellent mechanical and electrical properties. The in-vitro experiments indicate that the 3D-CSNFs could significantly enhance the growth and proliferation of hMSCs as well as their cellular collagen expression. Morphological studies



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showed that the 3D-CSNFs promote better support for the penetration, adhesion and interactions between the cells and the nanofibers compared to the 2D-CSNFs. In addition, hMSCs were cultured under electrical stimulations, which indicated that cells showed much better spread and proliferation compared to no electrical stimulation. We believe that the newly obtained 3D-CSNFs have great potentials for biomedical applications, such as the 3D bio-scaffold with electrical stimulation, bio-sensors, and conductive/dynamic cell systems for tissue engineering applications. AUTHOR INFORMATION Dr. L. Jin, J. B. Huang, 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: (86)394-8178518; Tel: (86)394-8178996 Prof. D. C. Wu* Q. W. Xu, S. Kuddannaya, C. Li, Prof. Y. L. Zhang* ‡

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

Nanyang Avenue, Singapore 639798, Singapore E-mail:[email protected] Dr. S. Y. Wu ⊥

Department of Prosthodontics, Guanghua School of Stomatology, Hospital of Stomatology,

Sun Yat-sen University, Guangzhou, 510055, P. R. China

Supporting Information


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Supporting Information is available: This material is available free of charge via the Internet at http://pubs.acs.org. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Grant No: 21404124 and 51572303), the project of Henan Province Key Discipline of Applied Chemistry (No: 201218692), and Program for Innovative Research Team (in Science and Technology) in University of Henan Province (14IRTSTHN009), and the program of Innovative Talent (in Science and Technology) in University of Henan Province (17HASTIT007). Y. Z. acknowledges the Tier-1 Academic Research Funds from the Singapore Ministry of Education (RGT 30/13) and A*STAR AOP project (1223600005). REFERENCES

(1) Huang, Z.; Newcomb, C. J.; Lei, Y.; Zhou, Y.; Bornstein, P.; Amendt, B. a.; Stupp, S. I.; Snead, M. L. Bioactive Nanofibers Enable the Identification of Thrombospondin as a Key Player in Enamel Regeneration. Biomaterials 2015, 61, 216-228. (2) Li, L.; Zhou, G.; Wang, Y.; Yang, G.; Ding, S.; Zhou, S. Controlled Dual Delivery of BMP-2 and Dexamethasone by Nanoparticle-Embedded Electrospun Nanofibers for the Efficient Repair of Critical-Sized Rat Calvarial Defect. Biomaterials 2015, 37, 218-229. (3) Tokarev, A.; Trotsenko, O.; Griffiths, I. M.; Stone, H. A.; Minko, S. Magnetospinning of Nano- and Microfibers. Adv. Mater. 2015, 27 (23), 3560-3565. (4) Liu, W.; Lipner, J.; Moran, C. H.; Feng, L.; Li, X.; Thomopoulos, S.; Xia, Y. Generation of Electrospun Nanofibers with Controllable Degrees of Crimping Through a Simple, Plasticizer-Based Treatment. Adv. Mater. 2015, 27 (16), 2583-2588. (5) Blum, A. P.; Kammeyer, J. K.; Rush, A. M.; Callmann, C. E.; Hahn, M. E.; Gianneschi, N. C. Stimuli-Responsive Nanomaterials for Biomedical Applications. J. Am. Chem. Soc. 2015, 137 (6), 2140-2154.



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Figure 1. Schematic representation of steps involved in the fabrication of conductive 3D-CSNFs. (I) Electrospinning nanofibers into DI water solution with FeCl3 and Pyrrole, (II) The pyrrole was polymerized under ultrasonic treatment, (III) 3D-CSNFs prepared by freeze drying.


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Figure 2. Photographic images of 2D-CSNFs (A) and 3D-CSNFs (B) before (left) and after PPy layer coating (right).



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Figure 3. SEM images of the 2D-CSNFs (A) and 3D-CSNFs (B) High magnification SEM image (C) and TEM image of 3D-CSNFs (D).


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Figure 4. Raman spectrum (A), XRD pattern (B), cyclic voltammograms (C) and strain-stress curve (D) of the 3D-CSNFs.



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Figure 5. Fluorescent 3D confocal images of hMSCs cultured for 5 days on the 2D-CSNFs and 3D-CSNFs.


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Figure 6. Morphology of hMSCs cultured 5 days on the 2D-CSNFs (A) and 3D-CSNFs (B). Attachment and proliferation of hMSCs cultured on the 2D-CSNFs and 3D-CSNFs measured during various successive incubation periods (C), and comparison of collagen content after 7 days culture (D). Data are mean ±SD, n = 3, *p < 0.05.



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Figure 7. Schematic of the bioreactor for hMSCs electrical stimulation.


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Figure 8. Fluorescent confocal images of hMSCs cultured for 4 days with/without electrical stimulation condition in 3D-CSNFs.



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Figure 9. Proliferation of hMSCs cultured for 7 days with/without electrical stimulation condition in 3D-CSNFs.


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For Table of Contents Use Only

Synergistic

Effects

of

Conductive

3D

Nanofibrous

Micro-environments and Electrical Stimulation on the Viability and Proliferation of Mesenchymal Stem Cells Lin Jin,†, ‡ Qinwei Xu,‡ Shuyi Wu,⊥ Shreyas Kuddannaya, ‡ Cheng Li, ‡ Jingbin Huang, † Yilei Zhang,‡* Zhenling Wang†*


Synergistic Effects of Conductive Three-Dimensional Nanofibrous

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