- Email: [email protected]
nanowire micropattern-based sensor for neural stem cell differentiation
Accepted Manuscript Title: Conductive Hydrogel/Nanowire Micropattern-based Sensor for Neural Stem Cell Differentiation Authors: Jong Min Lee, Joo Yoon Moon, Tae Hyun Kim, Seung Won Lee, Christian D. Ahrberg, Bong Geun Chung PII: DOI: Reference:
S0925-4005(17)32282-7 https://doi.org/10.1016/j.snb.2017.11.151 SNB 23645
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
9-5-2017 13-11-2017 24-11-2017
Please cite this article as: Jong Min Lee, Joo Yoon Moon, Tae Hyun Kim, Seung Won Lee, Christian D.Ahrberg, Bong Geun Chung, Conductive Hydrogel/Nanowire Micropattern-based Sensor for Neural Stem Cell Differentiation, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.11.151 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Conductive Hydrogel/Nanowire Micropattern-based Sensor for Neural Stem Cell Differentiation Jong Min Lee1,†, Joo Yoon Moon2,†, Tae Hyun Kim1, Seung Won Lee1, Christian D. Ahrberg1, Bong Geun Chung1,* Department of Mechanical Engineering, Sogang University, Seoul, Republic of Korea, 04107
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Department of Biomedical Engineering, Sogang University, Seoul, Republic of Korea, 04107
†
These authors contributed equally to this work
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* Corresponding author:
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Prof. Bong Geun Chung
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Sogang University, Seoul, Republic of Korea
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Department of Mechanical Engineering
Tel: 82-2-705-8823
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Fax: 82-2-712-0799
Highlights
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Email: [email protected]
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This manuscript describes the conductive hydrogel/silver nanowire (AgNW) composite micropattern-based sensor for neural stem cell differentiation and guidance of neurite outgrowth. The ridge micropatterns made from the composite PEG hydrogel/AgNW materials were fabricated on a flexible polyethylene terephthalate (PET) film. Through the
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combination of these composite materials and the ridge micropatterns, the outgrowth of neurites was efficiently guided in a controlled manner. Therefore, this conductive PEG hydrogel/AgNW micropattern-based sensor could potentially allow future use of the flexible device as an implant for neural stem cell therapy applications.
Abstract One of the main challenges regarding treatment of neurodegenerative diseases in the central nervous system (CNS) lies in the inability of neurons to undergo mitosis. Stem cell therapy could provide a possible solution to treat the neurodegenerative diseases. Here, we developed a conductive polyethylene glycol (PEG) hydrogel/silver nanowire (AgNW) composite micropattern-based sensor to direct differentiation of neuronal stem cells (NSCs) and guidance of neurite outgrowth. The ridge micropatterns made from the composite PEG hydrogel/AgNW materials were fabricated on a flexible
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polyethylene terephthalate (PET) film. Through the combination of these composite materials and the
ridge micropatterns, the outgrowth of neurites could be efficiently guided in a controlled manner.
Therefore, this conductive PEG hydrogel/AgNW micropattern-based sensor could potentially allow
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future use of the flexible device as an implant for NSC therapy applications.
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Keywords: Micropattern, Nanowire, Electrical stimuli, Sensor, Neural stem cell differentiation
1. Introduction Despite of the significant improvements achieved in the last decades, diseases affecting the nervous system like Alzheimer, Parkinson, and Huntington’s disease are still not curable [1, 2]. Likewise damage to the peripheral nervous system (PNS) or central nervous system (CNS) is not reversible due to the inability of neurons to undergo mitosis [3]. Although function can be restored with autographs in the PNS, these lead to a loss of function in the donor site, necessarily require several operations, and have a success rate of 80% [3]. Thus, researchers are developing new generations of implants
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with novel materials that should provide stability during nerve regeneration while being
biodegradable [4]. In 1995, McDonald et al. have shown that functional improvements can be
obtained through transplantation of neuronal cells differentiated from mouse embryonic stem cells
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into a damaged spinal cord [5]. As the rodent CNS is similar to the human one [6], this has sparked
the hope of curing physical damage of the CNS and PNS through stem cell therapies. Moreover, stem cell therapy might be able to provide cures for neural degenerative diseases. Currently, the main
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challenges faced in stem cell therapy are directing stem cell differentiation into the desired target
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phenotypes and spatial guidance of cell growth in a homogeneous manner [7]. Stem cell differentiation can be influenced by a variety of factors, such as chemical transcription
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factors [8, 9], chemogradients [10], and physical/mechanical factors [11]. In particular, the electrical
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stimuli can be used to guide stem cell differentiation towards a neural fate [12]. The microelectromechanical system (MEMS) provides an good method to apply the electrical fields [13]. Through electrical stimuli, it is not only possible to influence stem cell differentiation but also
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possible to direct neurite outgrowth, as was previously demonstrated guiding neurites into a hollow silicone implant [4]. In addition, faster growth rates and neurite migration towards the cathode can be
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observed when applying electrical stimuli [14, 15]. There are two different approaches for obtaining a well-defined and homogenous electrical field in the growth substrate. One option is the integration of conductive nanomaterials into the growth substrate, as was previously performed with gold
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nanoparticles [16]. The other option is to create the growth substrate from the conductive material itself. Previously, this was made by spinning nanofibers from conductive polymers [17], or using carbon nanotubes (CNT) forming two-dimensional (2D) conductive scaffolds [18, 19]. Threedimensional (3D) graphene foam was also used as a conductive scaffold, as they reassemble in vitro
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conditions better than 2D scaffolds [20]. Besides, the researchers have also varied the physical and mechanical properties of the substrate. Hydrogels are particularly interesting for this task, as their elastic modulus can be varied over several orders of magnitude [21], while providing a high permeability to water, oxygen, and water soluble metabolites [22, 23]. Thus, polyethylene glycol (PEG) hydrogels have been used for neural differentiation [24] and creation of 3D neuronal networks [25]. Biodegradable gelatin-hydroxyphenylpropionic acid hydrogels have been suggested as an injectable implant for stem cell therapy [26]. Furthermore, neurite outgrowth can be guided by
physical features on the surface of the substrate. This was demonstrated using a grooved poly(dimethylsiloxane) (PDMS) structure [27], the growth rate of neurites and stem cell differentiation efficiency could be increased by applying mechanical stress to these structures [28]. Feasibility of using patterned substrates as implants was also demonstrated by Hsu et al. [29]. Here, we demonstrate a novel approach combining a micropatterned PEG hydrogel substrate with silver nanowire (AgNW) to increase the conductivity of the substrate. Electrical stimuli applied to the substrate to enhance neural stem cell (NSC) differentiation and guide neurite outgrowth. To increase
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the conductivity of the material and provide a homogenous electrical field, AgNWs are integrated into
the PEG hydrogel matrix. To further guide neurite outgrowth, a series of parallel microridges are created from the hybrid hydrogel material. This, to our knowledge, is the first report of combining
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electrical stimuli and physical micropatterns containing AgNWs in one device for neurite guidance and NSC-derived neuronal differentiation. Therefore, we can achieve synergistic effects and obtain a more efficient guidance of neurites and a higher rate of neurite outgrowth compared to using electrical
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stimuli or micropatterns on their own.
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2. Material and methods
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2.1 Fabrication of PEG hydrogel/AgNW micropatterns
PDMS stamps for the fabrication of the PEG hydrogel/AgNW micropatterns were produced in a
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standard soft lithography process, as previously described.[24] Briefly, photomasks were designed using Autocad (Autodesk, USA) and reproduced in SU8-50 (Microchem Corp., USA) on silicon wafers (Wangxiang Silicon-Peak Electronics, China), a details for the fabrication process can be
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found in supplemental materials (Table S1). PDMS (10:1 Monomer: curing Agent, Microchem Corp., USA) was poured onto the silicon masters and cured in an oven at 85°C for one hour. The cured
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PDMS was carefully peeled from the silicon wafer and cut to size using a surgical blade, resulting in the PDMS stamps. To produce the PEG hydrogel/AgNW micropatterns, the PDMS stamps were placed onto a flexible PET film with a thickness of 100µm (Filmbank, Korea) and a precursor solution was injected in the gap between the PDMS stamp and the PET film. The precursor consisted
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of 10µL silver nanowire solution (Nanopyxis Co. Ltd., Korea), 100µL PEG-dimethacrylate (1000Da, Sigma Aldrich, USA), and 1 w/v% of the photoinitiator 2-hydroxy-2-methyl propiophenone (SigmaAldrich, USA). The hydrogel was polymerized in a radical chain growth reaction by illuminating for 30 seconds using ultraviolet light (320-500nm, Omni Cures Series 1500 curing station, EXFO, Canada). Finally, the PDMS stamp was carefully peeled off, leaving the PEG hydrogel/AgNW micropatterns on the PET film. PEG hydrogel/AgNW micropatterns were coated with a solution
containing 100µg/mL poly-L-ornithine (PLO, Sigma Sigma-Aldrich, USA) and 5µg/mL laminin (Sigma-Aldrich, USA) for one day to achieve a better attachment of the neurospheres to the hydrogel.
2.2 Characterization of composite hydrogel material Surface morphology of the pure PEG hydrogel, AgNWs, and PEG hydrogel/AgNW composite material were analyzed using SEM microscopy (JSM-7100f, Jeol USA, USA). Further mechanical
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properties of the pure PEG hydrogel and PEG hydrogel/AgNW composite material were tested. For this, disk of the materials were prepared by pipetting the prepolymer solution into a PDMS mold
(8mm diameter, 2mm height) and were subsequently polymerized. The compressive modulus was
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tested using a CT3 Texture Analyzer (Brookfield Engineering Laboratory, USA) equipped with a 4500g load cell (Brookfield Engineering Laboratory, USA). A probe diameter of 38mm was used for compressing the sample disks at a rate of 0.05mm/s. Stress-strain curves were recorded and
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compressive modules determined from the slope of a linear regression in the range of 5 to 15% strain. For comparison, gelatin methacrylate (GelMA, Sigma-Aldrich, USA), and matrigel (Gibco, USA)
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hydrogels were also tested for their mechanical properties using the same methods. Lastly, stress-
2.3 Simulation of electrical behavior
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strain curves of the PET film and the PEG hydrogel/AgNW micropatterns on PET film were analyzed.
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Prior to experiments, the electrical behavior of the PEG hydrogel/AgNW micropatterns was simulated using a Comsol Multiphysics 4.4 (Comsol Inc., USA) model. For the models, square modulated
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voltages with a frequency of 139µHz and magnitude of 5, 10, and 20V were chosen as used in
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experiments. The surface current density (𝑗⃗)) was then calculated using the following equation: 𝐽⃗ = 𝜎𝐸⃗⃗ =
𝐸⃗⃗ 𝜌
Where 𝐸⃗⃗ is the electric field in vector form, and σ is the conductivity of the material which can be
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expressed as the inverse of the resistivity (ρ). The electrical conductivity of water, and copper, which was used for the two electrodes, was taken the Comsol material library. For the other materials, no values were available in the material library, so values were taken from literature for PET [30], PEG [31], and AgNW [32]. As the conductivity of PEG hydrogel/AgNW composite materials is similar to bulk AgNWs [33] and the conductivity of AgNW was used.
2.4 NSC culture and neural differentiation NSC cells were extracted from cortex tissues of E12 C57BL/6 mice (Daehan Biolink, Korea). The cells were incubated with Accutase (Innovation Cell Technology, USA) at 37˚C for 50 minutes before growing them as neurospheres on ultra-low attachment surfaces for 4 days in DMEM/F12 medium (Gibco, USA) containing 20ng/mL basic fibroblast growth factor (bFGF) (R&D Systems, USA), 20ng/mL epidermal growth factor (EGF) (Invitrogen, USA), 1% N-2 Supplement, 2% B27 supplements, and 1% penicillin-streptomycin (Gibco, USA). The neurospheres were harvested and
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suspended in 100µL of the same medium and afterwards seeded onto PEG hydrogel/AgNW
micropatterns on the flexible PET film. After incubation for one day, a further 3mL of medium were
added. The electrical stimuli were then applied using a function generator (AFG1062, Tektronix, USA)
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and copper electrodes. Output voltages were increased to 5, 10, and 20V using a voltage amplifier
(HSA4011, NF Co, Japan). All voltages were modulated to give a single pulse every 2 hours (139µHz) for a total duration of 6 days. Control experiments were conducted on PET film submerged in culture
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medium, with application of identical electrical stimuli.
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2.5 Immunochemistry
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NSC differentiation was confirmed by an antibody staining reaction. In a first step, the cells on the PEG hydrogel/AgNW micropatterns were treated with 4% paraformaldehyde (YMS Korea, Korea) for 30 minutes and were subsequently permeabilized using 1% Triton X-100 (Sigma-Aldrich, USA)
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in phosphate-buffered saline (PBS, Sigma-Aldrich, USA). Non-specific protein binding was reduced through incubation with bovine serum albumin (BSA, 1 W/W%, Sigma-Aldrich, USA) in PBS at
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room temperature for 2 hours. The cells were then incubated with anti-neural class III primary antibodies, such as Tuj1 (Stem Cell Technology, Canada) or nestin (Abcam, UK) or MAP2 (Abcam, UK), at 4°C overnight. They were rinsed with PBS buffer and were subsequently incubated overnight
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at 4˚C with Alexa Flour 488-conjugated secondary antibody (Tuj1, Invitrogen, USA) and Alexa Flour 594-conjugated secondary antibody (Map2/nestin, Abcam, UK). Finally, nuclei were stained by a solution of 0.1 g/mL 4’,6-diamidino-2-phenylindole (DAPI, Thermo Fisher, USA) in PBS buffer for 30 min at room temperature. After rinsing PEG hydrogel/AgNW micropatterns with PBS buffer,
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fluorescent images were taken using a confocal laser-scanning microscope (LSM710, Carl Zeiss, Germany). Image J (National Institute of Health, USA) was used to measure the direction of neurite outgrowth, which was classified into eight different directions.
3. Results and discussion
3.1 Fabrication and characterization of PEG hydrogel/AgNW micropatterns PEG hydrogel/AgNW micropatterns on a flexible PET film were made from PDMS molds (Fig. 1A). 200µm ridge microstructures were made in a controllable and reproducible manner (Fig. 1B,C). Afterwards the PEG hydrogel/AgNW micropatterns were submerged in the cell growth medium and a function generator-mediated voltage subsequently applied after the cell seeding. Material properties were observed by scanning electron microscopy (SEM) of the PEG hydrogel, AgNW, and the composite 10% PEG hydrogel/AgNW material (Fig. 2). The 10% PEG hydrogel displayed a series of
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crests and valleys on its surface (Fig. 2A). The AgNWs, as expected, showed long and thin wires (Fig. 2B). The PEG hydrogel/AgNW composite material displayed a surface morphology similar to the
PEG hydrogel with ridges and valleys of similar dimensions as the pure hydrogel (Fig. 2C). Within
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the hydrogel, individual AgNW can be observed in the bulk hydrogel. As expected, the AgNWs are randomly distributed within the hydrogel. Randomly distributed AgNWs allow for the formation of conductive networks, as previously shown [34]. However, aligning AgNWs leads to a higher
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conductivity at the same nanowire concentration [35], which could allow reducing the material costs of the device. Additionally, X-ray spectroscopy was used to analyze the elemental composition of the
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composite material (Fig. 2D). Unsurprisingly, the two most abundant elements found were carbon and
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oxygen in nearly the ratio expected from the hydrogel. Additionally, almost 5wt% silver was found by elemental X-ray spectroscopy, which was higher than the expected value of 2wt%. As only a small
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sample, containing AgNW, was chosen for X-ray spectroscopy, the content of AgNW was not representative for the bulk material. Hence, the measured content of silver nanowires was higher than
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the expected value. Lastly, small amounts of sodium and chloride were found. These originated from salts in the hydrogel precursor in the synthesis of the hydrogel. Through the drying of the material prior to SEM analysis, the concentration was increased compared to the hydrated form of the
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hydrogels. We also characterized the mechanical properties of the PEG hydrogel/AgNW materials and their properties compared to other hydrogels (Fig. 3). Although one would expect an altered
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mechanical behavior of the hydrogel after the addition of AgNWs, the stress-strain curves for PEG and the PEG hydrogel/AgNW composite materials were similar and their compressive moduli were not significantly different. It is due to the amount of AgNWs (~0.2wt%). Although the desired electrical properties are achieved, The amount of AgNW (~0.2wt%) is too small to lead to a
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measureable difference in mechanical properties. As a result, we confirmed that the addition of low concentrations of AgNWs to PEG hydrogels did not significantly alter their mechanical properties. In contrast, the compressive modulus of the matrigel was significantly lower than that of PEG hydrogel/AgNW composite materials (Fig. 3B). Furthermore, we analyzed stress-strain curves for tension tests between PEG hydrogel/AgNW micropatterns on PET film and pure PET film (Fig. S1). As expected, it showed that they were almost identical. The addition of the supporting PET film can
lead to suitable mechanical properties. However, the PET film needs to be replaced by a biodegradable material to obtain a fully degradable neural implant or graft.
3.2 Simulation of electrical behavior Before conducting experiments, the electrical behavior of PEG hydrogel/AgNW micropatterns was simulated using Comsol Multiphysics software (Fig. 4). Firstly, the current density within the PEG
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hydrogel/AgNW micropatterns was simulated (Fig. 4). From the simulation, it can be seen that when applying a voltage the current density within the PEG hydrogel/AgNW structure is higher than the
current density of the surrounding medium and the supporting PET film. Simulations of the PEG
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hydrogel/AgNW microstructure parallel to the applied voltage have shown that the current density
within the structures would be less homogeneous. Hence, to enhance homogeneity, it was decided to use the PEG hydrogel/AgNW microstructure perpendicular to the applied voltage. The model further
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suggests that the simulated current density within the individual ridges of the PEG hydrogel/AgNW is inhomogeneous. This effect is related to the mesh size of the Comsol model and numerical
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instabilities during the simulation. In experiments, we expect a homogeneous current distribution
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within the individual ridges of the microstructure. Secondly, the average current at the wall in the different materials was simulated (Fig. 4C). It was observed that PET and PEG showed lower currents
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than AgNW due to the lower conductivity of the materials. For the PEG hydrogel/AgNW material, a current similar to AgNWs can be expected due to similar conductivities [33], showing the feasibility
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of the material in the conduction of electrical stimuli. Furthermore, the dependency of the current inside the ridges on the applied voltage was simulated (Fig. 4D). As expected from Ohm’s law, a liner
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relationship between current and applied voltage was found.
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3.3 NSC-derived neural differentiation To optimize the dimension of PEG hydrogel/AgNW micropatterns, we investigated various widths of PEG hydrogel/AgNW micropatterns without applying a voltage (Fig. 5). Choosing a width of the
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micropatterns smaller than the diameter of the neurospheres can lead to neurosphere growing on several microridges simultaneously. Through the spatial proximity of neurospheres on neighboring ridges, the fusion of several neurospheres was observed (Fig. 5A). If a PEG hydrogel/AgNW micropattern width wider than the diameter of the neurospheres is used, the guiding efficiency of neurites cultured in the micropatterns may be lost. This is observable by the radial outgrowth of neurites (Fig. 5C,D). Therefore, we used PEG hydrogel/AgNW micropatterns with 200µm width, which was similar to the diameter of neurospheres (Fig. 5B), showing the best compromise between
neurite guiding and neurosphere seeding efficiency. To determine the differentiation of NSCs into neurons in the PEG hydrogel/AgNW micropatterns, the neural markers (e.g., Tuj1, MAP2, and nestin) were assessed by specific immunostaining with fluorescently labeled antibodies (Fig. 6). From the fluorescent microscopy images, it was observed that a large number of cells differentiated into Tuj1postive neuronal cells. To gain further information about NSC differentiation behaviors, we used Map2 and nestin biomarker (Fig. S2). From the fluorescence microscopy images, we observed that a number of NSCs differentiated into MAP2-positive mature neuronal cells, while a small number of
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cells are nestin-positive neural progenitor cells. Furthermore, the outgrowth of neurites mediated from the electrical stimuli was observed. However, the strongest growth of neurites, both by number and length, was observed when applying intermediated voltages of 5 and 10V (Fig. 6 B,C,F,G). Longer
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neurites can be observed in the PEG hydrogel/AgNW micropatterns (Fig. 6G) compared to control experiments at the same voltage (Fig. 6C). Furthermore, while the direction of neurite growth appears to be random in control experiments, neurite outgrowth appears to follow the PEG hydrogel/AgNW
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micropatterns.
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3.4 Analysis of neurite outgrowth
The neurite outgrowth was measured with Image J and analyzed regarding neurite number and length
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(Fig. 7) and the growth direction (Fig. 8). In experiments conducted with the PEG hydrogel/AgNW micropattern, the average length of neurites was increasing with increasing applied voltages (Fig. 7A).
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Furthermore, the number of neurites increased with the applied voltage (Fig. 7B). Application of low voltages of 10V significantly increases the length of neurites compared to control experiments, while the number of neurites also increases compared to no voltage control experiments, confirming results
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by Patel et al. [36]. However, when applying a higher voltage of 20V, neither neurite length nor neurite number is significantly increased compared to control experiments without electrical stimuli.
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Similarly, Yamada et al. observed a lower differentiation efficiency at voltages of 20V compared to 15V [12]. The mechanism for this is still not fully understood [37]. A possible reason is that low electric fields that present in wound healing, affect the cell steering mechanism, while higher electric fields (>2V/cm) also stimulate overall cell motor activity [38]. In addition to the length and number of
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neurites, the direction of neurite outgrowth was also analyzed (Fig. 8). In control experiments, neurites could be observed growing in all directions, with a fairly even distribution (Fig. 8A). In literature a tendency of neurites to grow towards the cathode was observed [36]. However in our experiments this tendency was not observed. A possible reason for this might be that in our experiments the frequency of stimulation (f=139µHz) is four orders of magnitude lower than used in previous studies (f=1Hz) [36], which observed growth towards the cathode. In contrast, neurite growth orients along the ridges of the substrate when an electrical field is applied (Fig. 8B). In
experiments with no applied voltage, the direction of neurite outgrowth appears to be random, as observed in control experiments. Nevertheless, when a voltage of 5 or 10V is applied, the outgrowth of neurites orients itself along the ridges of the PEG hydrogel/AgNW micropattern. In direct comparison of the control experiment and the PEG hydrogel/AgNW micropattern at 10V, it can be seen that on the microstructure 60% of neurites follow the structure in their growth direction, while only 20% of neurites grow in the direction of the electrode for the control experiments (Fig. 8C). This superior performance of the PEG hydrogel/AgNW micropattern could be attributed to two different
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factors. Firstly, the ridges of the material impose spatial limitations to the growth of neurites, especially for neurites close to the edge of a ridge. This effect would be more visible with longer neurites, as observed in 10V experiments, as they are more likely to encounter an edge of the ridges.
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Secondly, AgNW in the composite material might aid as a guide to the outgrowth of neurites.
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4. Conclusions
We presented a conductive PEG hydrogel/AgNW composite micropattern-based sensor that was used
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for NSC differentiation into neurons under the application of electrical stimuli. Microstructures were
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formed from the composite material with the aid of a PDMS mold, through which neurite outgrowth could be guided. Simulation of the electrical behavior of the composite material and structure
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demonstrated the advantages of the PEG hydrogel/AgNW composite material compared to conventional hydrogels. Therefore, our PEG hydrogel/AgNW composite micropattern-based sensor
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NSC therapy applications.
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on a flexible, supporting PET film could allow the potential future use as a neural implant device for
Acknowledgements
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This research was supported by BioNano Health-Guard Research Center funded by the Ministry of
Science
and
ICT
(MSIT)
of
Korea
as
Global
Frontier
Project
(grant
number
H-
GUARD_2014M3A6B2060503), Republic of Korea. This work was supported by the National Foundation
(NRF)
of
Korea
grant
funded
by
the
MSIT
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Research
(Grant
number
2016R1A6A1A03012845). This research was also supported by Basic Science Research Program through the NRF funded by the Ministry of Education (Grant number 2016R1A6A3A11931838). Authors appreciate Prof. Sun Woong in Korea University to kindly provide NSCs.
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[19] Y.J. Huang, H.C. Wu, N.H. Tai, T.W. Wang, Carbon Nanotube Rope with Electrical Stimulation Promotes the Differentiation and Maturity of Neural Stem Cells, Small 8 (2012) 2869-2877. [20] N. Li, Q. Zhang, S. Gao, Q. Song, R. Huang, L. Wang, L. Liu, J. Dai, M. Tang, G. Cheng, Threedimensional graphene foam as a biocompatible and conductive scaffold for neural stem cells, Sci. Rep. 3 (2013) 1604. [21] A. Banerjee, M. Arha, S. Choudhary, R.S. Ashton, S.R. Bhatia, D.V. Schaffer, R.S. Kane, The influence of hydrogel modulus on the proliferation and differentiation of encapsulated neural stem cells, Biomaterials 30 (2009) 4695-4699. [22] S. Cosson, M.P. Lutolf, Hydrogel microfluidics for the patterning of pluripotent stem cells, Sci. Rep. 4 (2014) 4462. [23] B.G. Chung, K.H. Lee, A. Khademhosseini, S.H. Lee, Microfluidic fabrication of microengineered hydrogels and their application in tissue engineering, Lab Chip 12 (2012) 45-59. [24] J.H. Bae, J.M. Lee, B.G. Chung, Hydrogel-encapsulated 3D microwell array for neuronal differentiation, Biomed. Mater. 11 (2016) 015019. [25] N.R. Wevers, R. van Vught, K.J. Wilschut, A. Nicolas, C. Chiang, H.L. Lanz, S.J. Trietsch, J. Joore, P. Vulto, High-throughput compound evaluation on 3D networks of neurons and glia in a microfluidic platform, Sci. Rep. 6 (2016) 38856. [26] L.S. Wang, J.E. Chung, P. Pui-Yik Chan, M. Kurisawa, Injectable biodegradable hydrogels with tunable mechanical properties for the stimulation of neurogenesic differentiation of human mesenchymal stem cells in 3D culture, Biomaterials 31 (2010) 1148-1157. [27] A. Béduer, C. Vieu, F. Arnauduc, J.C. Sol, I. Loubinoux, L. Vaysse, Engineering of adult human neural stem cells differentiation through surface micropatterning, Biomaterials 33 (2012) 504-514. [28] Y.J. Chang, C.J. Tsai, F.G. Tseng, T.J. Chen, T.W. Wang, Micropatterned stretching system for the investigation of mechanical tension on neural stem cells behavior, Nanomed. Nanotechnol. Biol. Med. 9 (2013) 345-355. [29] S.h. Hsu, C.H. Su, I.M. Chiu, A Novel Approach to Align Adult Neural Stem Cells on Micropatterned Conduits for Peripheral Nerve Regeneration: A Feasibility Study, Artif. Organs. 33 (2009) 26-35. [30] G. Hu, C. Zhao, S. Zhang, M. Yang, Z. Wang, Low percolation thresholds of electrical conductivity and rheology in poly(ethylene terephthalate) through the networks of multi-walled carbon nanotubes, Polymer 47 (2006) 480-488. [31] M.V. Murugendrappa, S. Khasim, M.V.N.A. Prasad, Conductivity and DSC studies of poly(ethylene glycol) and its salt complexes, Indian J. Eng. Mater. Sci. 7 (2000) 456-458. [32] Y. Sun, Y. Yin, B.T. Mayers, T. Herricks, Y. Xia, Uniform Silver Nanowires Synthesis by Reducing AgNO3 with Ethylene Glycol in the Presence of Seeds and Poly(Vinyl Pyrrolidone), Chem. Mater. 14 (2002) 4736-4745. [33] L. Song, A.C. Myers, J.J. Adams, Y. Zhu, Stretchable and Reversibly Deformable Radio Frequency Antennas Based on Silver Nanowires, ACS Appl. Mater. Interfaces. 6 (2014) 4248-4253. [34] Y. Ko, J. Kim, D. Kim, Y. Yamauchi, J.H. Kim, J. You, A Simple Silver Nanowire Patterning Method Based on Poly(Ethylene Glycol) Photolithography and Its Application for Soft Electronics, Sci. Rep. 7 (2017) 2282. [35] W.R. Small, V.N. Paunov, Dielectrophoretic fabrication of electrically anisotropic hydrogels with bio-functionalised silver nanowires, J. Mater. Chem. B. 1 (2013) 5798-5805. [36] N. Patel, M. Poo, Orientation of neurite growth by extracellular electric fields, J. Neurosci. 2 (1982) 483-496. [37] F. Pires, Q. Ferreira, C.A.V. Rodrigues, J. Morgado, F.C. Ferreira, Neural stem cell differentiation by electrical stimulation using a cross-linked PEDOT substrate: Expanding the use of
biocompatible conjugated conductive polymers for neural tissue engineering, Biochim. Biophys. Acta. 1850 (2015) 1158-1168. [38] X. Li, J. Kolega, Effects of Direct Current Electric Fields on Cell Migration and Actin Filament Distribution in Bovine Vascular Endothelial Cells, J. Vasc. Res. 39 (2002) 391-404.
Biographies
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Jong Min Lee received his B.S. degree in Biochemical Engineering from Korea Polytechnic University, Korea in 2010, respectively. In 2016, he received his M.S. and Ph.D. degree in Mechanical Engineering, both from Sogang University, Korea. Currently, he is a research professor for the Department of Mechanical Engineering at Sogang University, Seoul, Korea. His current research interests are development of microfluidic sensing devices for healthcare and medical applications. Joo Yoon Moon received his B.S. degree in Biomedical Engineering at Kyung Hee University, Korea in 2015 and received his M.S. from Sogang University, Korea (2017). His research interests are microfluidics and stem cells for diagnosis and treatment of disease.
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Tae Hyun Kim received the B.S. degree from Seoul National University of Science and Technology in Mechanical System design engineering (2012). He received his M.S degree in Mechanical Engineering in 2015 at Sogang University, Korea. Currently, he is a Ph.D. degree candidate at Department of Mechanical Engineering in Sogang University. His research interests are modeling and simulation of multi-physics in bio-medical application.
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Seung Won Lee received his B.S. degree at Hanyang University with Material Engineering in 2000. In 2017, he received his M.S. at Department of Mechanical Engineering in Sogang University, Korea. Currently, he is a Ph.D. degree candidate at Department of Mechanical Engineering in Sogang University. He is also Senior Manager for Platform Technology Lab, SAIT, in Samsung Electronics, Korea. His current research interests are fabricating sensing device for electrical applications.
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Christian D. Ahrberg obtained his Bachelor and Master Degree in Chemical and Bioengineering in 2012. He worked at Bayer Technology Services in Leverkusen before starting his Ph.D. under supervision of Andreas Manz at the Mechatronics department of Saarland University in cooperation with KIST Europe, during which he worked on a portable real-time PCR device. Since graduating in 2016, he is currently a postdoctoral researcher at the BioNano Technology Lab in Sogang University, Seoul, Korea.
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Bong Geun Chung received the B.S. and M.S. degree from Hanyang University, Seoul, Korea. In 2007, he obtained Ph.D. degree from Materials Science Engineering in University of California Irvine, USA. After Ph.D. degree, he worked as a Postdoctoral Research Fellow and an Instructor in Harvard Medical School, USA. Currently, he is an Associate Professor at Department of Mechanical Engineering in Sogang University, Seoul, Korea. His main research interests are development of functional 3D Lab-on-a-Chips, Biosensors, and Nanomaterials to direct the stem cell and cancer fate.
Figure Legends Fig. 1. Schematic of the production of PEG hydrogel/AgNW micropattern-based sensor (A). Photograph of the PEG hydrogel/AgNW micropattern inside of the PDMS mold. PEG hydrogel/AgNW micropattern is stained with red dye. Scale bar is 1cm (B). Fluorescent microscopy image of PEG hydrogel/AgNW micropattern after removal of the PDMS mold. For illustration
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purposes the hydrogel is stained with fluorescein dye. Scale bar is 100µm (C).
Fig. 2. SEM images of the PEG hydrogel (A), AgNW (B), and PEG hydrogel/AgNW composite
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material (C). X-ray element spectroscopy of composite PEG hydrogel/AgNW composite material (D).
Fig. 3. Stress-strain curve of compression test of different hydrogel materials (A) and analysis of compressive modulus (B). The compressive modulus is significantly different between PEG
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hydrogel/AgNW composite material and matrigel (**p<0.01).
Fig. 4. Simulations of electrical behavior of the composite PEG hydrogel/AgNW micropattern.
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Schematic of experimental setup for applying electrical stimuli (A). Simulated current density in the micropattern with an applied voltage of 10V (B). Analysis of simulated average current for different
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materials at an applied voltage of 10V (C) and current for the PEG hydrogel/AgNW composite
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material at different applied voltages (D).
Fig. 5. Fluorescent microscopy images of NSCs on PEG hydrogel/AgNW micropatterns with 100µm
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(A), 200µm (B), 300µm (C), and 500µm (D) width. A green dye was used for Tuj1, red for nestin, and blue for DAPI.
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Fig. 6. Fluorescent microscopy images of NSCs after culture in control experiments (A-D) and PEG hydrogel/AgNW micropatterns (E-H). To facilitate NSC differentiation, various electric stimuli are applied. Cells are immunostained by TuJ1 (green) and cell nuclei are stained by DAPI (blue). Scale bars are 200µm.
Fig. 7. Analysis of average neurite length (A), average number of neurites (B) for NSCs cultured on the PEG hydrogel/AgNW micropattern.
Fig. 8. Rose diagrams indicating percentage of neurites growing in a certain direction in control experiments (A) and in experiment conducted on the PEG hydrogel/AgNW micropatterns (B). Rose diagram comparing the percentage of neurites growing in a certain direction of control and
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micropattern experiments (C). 0˚ is growth towards the top of the micropattern and 90˚ is growth towards the cathode, respectively. Growth was classified into eight different directions, each
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S0925-4005(17)32282-7 https://doi.org/10.1016/j.snb.2017.11.151 SNB 23645
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
9-5-2017 13-11-2017 24-11-2017
Please cite this article as: Jong Min Lee, Joo Yoon Moon, Tae Hyun Kim, Seung Won Lee, Christian D.Ahrberg, Bong Geun Chung, Conductive Hydrogel/Nanowire Micropattern-based Sensor for Neural Stem Cell Differentiation, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.11.151 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Conductive Hydrogel/Nanowire Micropattern-based Sensor for Neural Stem Cell Differentiation Jong Min Lee1,†, Joo Yoon Moon2,†, Tae Hyun Kim1, Seung Won Lee1, Christian D. Ahrberg1, Bong Geun Chung1,* Department of Mechanical Engineering, Sogang University, Seoul, Republic of Korea, 04107
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Department of Biomedical Engineering, Sogang University, Seoul, Republic of Korea, 04107
†
These authors contributed equally to this work
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* Corresponding author:
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Prof. Bong Geun Chung
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Sogang University, Seoul, Republic of Korea
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Department of Mechanical Engineering
Tel: 82-2-705-8823
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Fax: 82-2-712-0799
Highlights
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Email: [email protected]
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This manuscript describes the conductive hydrogel/silver nanowire (AgNW) composite micropattern-based sensor for neural stem cell differentiation and guidance of neurite outgrowth. The ridge micropatterns made from the composite PEG hydrogel/AgNW materials were fabricated on a flexible polyethylene terephthalate (PET) film. Through the
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combination of these composite materials and the ridge micropatterns, the outgrowth of neurites was efficiently guided in a controlled manner. Therefore, this conductive PEG hydrogel/AgNW micropattern-based sensor could potentially allow future use of the flexible device as an implant for neural stem cell therapy applications.
Abstract One of the main challenges regarding treatment of neurodegenerative diseases in the central nervous system (CNS) lies in the inability of neurons to undergo mitosis. Stem cell therapy could provide a possible solution to treat the neurodegenerative diseases. Here, we developed a conductive polyethylene glycol (PEG) hydrogel/silver nanowire (AgNW) composite micropattern-based sensor to direct differentiation of neuronal stem cells (NSCs) and guidance of neurite outgrowth. The ridge micropatterns made from the composite PEG hydrogel/AgNW materials were fabricated on a flexible
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polyethylene terephthalate (PET) film. Through the combination of these composite materials and the
ridge micropatterns, the outgrowth of neurites could be efficiently guided in a controlled manner.
Therefore, this conductive PEG hydrogel/AgNW micropattern-based sensor could potentially allow
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future use of the flexible device as an implant for NSC therapy applications.
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Keywords: Micropattern, Nanowire, Electrical stimuli, Sensor, Neural stem cell differentiation
1. Introduction Despite of the significant improvements achieved in the last decades, diseases affecting the nervous system like Alzheimer, Parkinson, and Huntington’s disease are still not curable [1, 2]. Likewise damage to the peripheral nervous system (PNS) or central nervous system (CNS) is not reversible due to the inability of neurons to undergo mitosis [3]. Although function can be restored with autographs in the PNS, these lead to a loss of function in the donor site, necessarily require several operations, and have a success rate of 80% [3]. Thus, researchers are developing new generations of implants
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with novel materials that should provide stability during nerve regeneration while being
biodegradable [4]. In 1995, McDonald et al. have shown that functional improvements can be
obtained through transplantation of neuronal cells differentiated from mouse embryonic stem cells
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into a damaged spinal cord [5]. As the rodent CNS is similar to the human one [6], this has sparked
the hope of curing physical damage of the CNS and PNS through stem cell therapies. Moreover, stem cell therapy might be able to provide cures for neural degenerative diseases. Currently, the main
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challenges faced in stem cell therapy are directing stem cell differentiation into the desired target
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phenotypes and spatial guidance of cell growth in a homogeneous manner [7]. Stem cell differentiation can be influenced by a variety of factors, such as chemical transcription
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factors [8, 9], chemogradients [10], and physical/mechanical factors [11]. In particular, the electrical
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stimuli can be used to guide stem cell differentiation towards a neural fate [12]. The microelectromechanical system (MEMS) provides an good method to apply the electrical fields [13]. Through electrical stimuli, it is not only possible to influence stem cell differentiation but also
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possible to direct neurite outgrowth, as was previously demonstrated guiding neurites into a hollow silicone implant [4]. In addition, faster growth rates and neurite migration towards the cathode can be
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observed when applying electrical stimuli [14, 15]. There are two different approaches for obtaining a well-defined and homogenous electrical field in the growth substrate. One option is the integration of conductive nanomaterials into the growth substrate, as was previously performed with gold
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nanoparticles [16]. The other option is to create the growth substrate from the conductive material itself. Previously, this was made by spinning nanofibers from conductive polymers [17], or using carbon nanotubes (CNT) forming two-dimensional (2D) conductive scaffolds [18, 19]. Threedimensional (3D) graphene foam was also used as a conductive scaffold, as they reassemble in vitro
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conditions better than 2D scaffolds [20]. Besides, the researchers have also varied the physical and mechanical properties of the substrate. Hydrogels are particularly interesting for this task, as their elastic modulus can be varied over several orders of magnitude [21], while providing a high permeability to water, oxygen, and water soluble metabolites [22, 23]. Thus, polyethylene glycol (PEG) hydrogels have been used for neural differentiation [24] and creation of 3D neuronal networks [25]. Biodegradable gelatin-hydroxyphenylpropionic acid hydrogels have been suggested as an injectable implant for stem cell therapy [26]. Furthermore, neurite outgrowth can be guided by
physical features on the surface of the substrate. This was demonstrated using a grooved poly(dimethylsiloxane) (PDMS) structure [27], the growth rate of neurites and stem cell differentiation efficiency could be increased by applying mechanical stress to these structures [28]. Feasibility of using patterned substrates as implants was also demonstrated by Hsu et al. [29]. Here, we demonstrate a novel approach combining a micropatterned PEG hydrogel substrate with silver nanowire (AgNW) to increase the conductivity of the substrate. Electrical stimuli applied to the substrate to enhance neural stem cell (NSC) differentiation and guide neurite outgrowth. To increase
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the conductivity of the material and provide a homogenous electrical field, AgNWs are integrated into
the PEG hydrogel matrix. To further guide neurite outgrowth, a series of parallel microridges are created from the hybrid hydrogel material. This, to our knowledge, is the first report of combining
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electrical stimuli and physical micropatterns containing AgNWs in one device for neurite guidance and NSC-derived neuronal differentiation. Therefore, we can achieve synergistic effects and obtain a more efficient guidance of neurites and a higher rate of neurite outgrowth compared to using electrical
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stimuli or micropatterns on their own.
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2. Material and methods
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2.1 Fabrication of PEG hydrogel/AgNW micropatterns
PDMS stamps for the fabrication of the PEG hydrogel/AgNW micropatterns were produced in a
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standard soft lithography process, as previously described.[24] Briefly, photomasks were designed using Autocad (Autodesk, USA) and reproduced in SU8-50 (Microchem Corp., USA) on silicon wafers (Wangxiang Silicon-Peak Electronics, China), a details for the fabrication process can be
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found in supplemental materials (Table S1). PDMS (10:1 Monomer: curing Agent, Microchem Corp., USA) was poured onto the silicon masters and cured in an oven at 85°C for one hour. The cured
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PDMS was carefully peeled from the silicon wafer and cut to size using a surgical blade, resulting in the PDMS stamps. To produce the PEG hydrogel/AgNW micropatterns, the PDMS stamps were placed onto a flexible PET film with a thickness of 100µm (Filmbank, Korea) and a precursor solution was injected in the gap between the PDMS stamp and the PET film. The precursor consisted
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of 10µL silver nanowire solution (Nanopyxis Co. Ltd., Korea), 100µL PEG-dimethacrylate (1000Da, Sigma Aldrich, USA), and 1 w/v% of the photoinitiator 2-hydroxy-2-methyl propiophenone (SigmaAldrich, USA). The hydrogel was polymerized in a radical chain growth reaction by illuminating for 30 seconds using ultraviolet light (320-500nm, Omni Cures Series 1500 curing station, EXFO, Canada). Finally, the PDMS stamp was carefully peeled off, leaving the PEG hydrogel/AgNW micropatterns on the PET film. PEG hydrogel/AgNW micropatterns were coated with a solution
containing 100µg/mL poly-L-ornithine (PLO, Sigma Sigma-Aldrich, USA) and 5µg/mL laminin (Sigma-Aldrich, USA) for one day to achieve a better attachment of the neurospheres to the hydrogel.
2.2 Characterization of composite hydrogel material Surface morphology of the pure PEG hydrogel, AgNWs, and PEG hydrogel/AgNW composite material were analyzed using SEM microscopy (JSM-7100f, Jeol USA, USA). Further mechanical
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properties of the pure PEG hydrogel and PEG hydrogel/AgNW composite material were tested. For this, disk of the materials were prepared by pipetting the prepolymer solution into a PDMS mold
(8mm diameter, 2mm height) and were subsequently polymerized. The compressive modulus was
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tested using a CT3 Texture Analyzer (Brookfield Engineering Laboratory, USA) equipped with a 4500g load cell (Brookfield Engineering Laboratory, USA). A probe diameter of 38mm was used for compressing the sample disks at a rate of 0.05mm/s. Stress-strain curves were recorded and
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compressive modules determined from the slope of a linear regression in the range of 5 to 15% strain. For comparison, gelatin methacrylate (GelMA, Sigma-Aldrich, USA), and matrigel (Gibco, USA)
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hydrogels were also tested for their mechanical properties using the same methods. Lastly, stress-
2.3 Simulation of electrical behavior
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strain curves of the PET film and the PEG hydrogel/AgNW micropatterns on PET film were analyzed.
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Prior to experiments, the electrical behavior of the PEG hydrogel/AgNW micropatterns was simulated using a Comsol Multiphysics 4.4 (Comsol Inc., USA) model. For the models, square modulated
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voltages with a frequency of 139µHz and magnitude of 5, 10, and 20V were chosen as used in
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experiments. The surface current density (𝑗⃗)) was then calculated using the following equation: 𝐽⃗ = 𝜎𝐸⃗⃗ =
𝐸⃗⃗ 𝜌
Where 𝐸⃗⃗ is the electric field in vector form, and σ is the conductivity of the material which can be
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expressed as the inverse of the resistivity (ρ). The electrical conductivity of water, and copper, which was used for the two electrodes, was taken the Comsol material library. For the other materials, no values were available in the material library, so values were taken from literature for PET [30], PEG [31], and AgNW [32]. As the conductivity of PEG hydrogel/AgNW composite materials is similar to bulk AgNWs [33] and the conductivity of AgNW was used.
2.4 NSC culture and neural differentiation NSC cells were extracted from cortex tissues of E12 C57BL/6 mice (Daehan Biolink, Korea). The cells were incubated with Accutase (Innovation Cell Technology, USA) at 37˚C for 50 minutes before growing them as neurospheres on ultra-low attachment surfaces for 4 days in DMEM/F12 medium (Gibco, USA) containing 20ng/mL basic fibroblast growth factor (bFGF) (R&D Systems, USA), 20ng/mL epidermal growth factor (EGF) (Invitrogen, USA), 1% N-2 Supplement, 2% B27 supplements, and 1% penicillin-streptomycin (Gibco, USA). The neurospheres were harvested and
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suspended in 100µL of the same medium and afterwards seeded onto PEG hydrogel/AgNW
micropatterns on the flexible PET film. After incubation for one day, a further 3mL of medium were
added. The electrical stimuli were then applied using a function generator (AFG1062, Tektronix, USA)
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and copper electrodes. Output voltages were increased to 5, 10, and 20V using a voltage amplifier
(HSA4011, NF Co, Japan). All voltages were modulated to give a single pulse every 2 hours (139µHz) for a total duration of 6 days. Control experiments were conducted on PET film submerged in culture
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medium, with application of identical electrical stimuli.
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2.5 Immunochemistry
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NSC differentiation was confirmed by an antibody staining reaction. In a first step, the cells on the PEG hydrogel/AgNW micropatterns were treated with 4% paraformaldehyde (YMS Korea, Korea) for 30 minutes and were subsequently permeabilized using 1% Triton X-100 (Sigma-Aldrich, USA)
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in phosphate-buffered saline (PBS, Sigma-Aldrich, USA). Non-specific protein binding was reduced through incubation with bovine serum albumin (BSA, 1 W/W%, Sigma-Aldrich, USA) in PBS at
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room temperature for 2 hours. The cells were then incubated with anti-neural class III primary antibodies, such as Tuj1 (Stem Cell Technology, Canada) or nestin (Abcam, UK) or MAP2 (Abcam, UK), at 4°C overnight. They were rinsed with PBS buffer and were subsequently incubated overnight
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at 4˚C with Alexa Flour 488-conjugated secondary antibody (Tuj1, Invitrogen, USA) and Alexa Flour 594-conjugated secondary antibody (Map2/nestin, Abcam, UK). Finally, nuclei were stained by a solution of 0.1 g/mL 4’,6-diamidino-2-phenylindole (DAPI, Thermo Fisher, USA) in PBS buffer for 30 min at room temperature. After rinsing PEG hydrogel/AgNW micropatterns with PBS buffer,
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fluorescent images were taken using a confocal laser-scanning microscope (LSM710, Carl Zeiss, Germany). Image J (National Institute of Health, USA) was used to measure the direction of neurite outgrowth, which was classified into eight different directions.
3. Results and discussion
3.1 Fabrication and characterization of PEG hydrogel/AgNW micropatterns PEG hydrogel/AgNW micropatterns on a flexible PET film were made from PDMS molds (Fig. 1A). 200µm ridge microstructures were made in a controllable and reproducible manner (Fig. 1B,C). Afterwards the PEG hydrogel/AgNW micropatterns were submerged in the cell growth medium and a function generator-mediated voltage subsequently applied after the cell seeding. Material properties were observed by scanning electron microscopy (SEM) of the PEG hydrogel, AgNW, and the composite 10% PEG hydrogel/AgNW material (Fig. 2). The 10% PEG hydrogel displayed a series of
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crests and valleys on its surface (Fig. 2A). The AgNWs, as expected, showed long and thin wires (Fig. 2B). The PEG hydrogel/AgNW composite material displayed a surface morphology similar to the
PEG hydrogel with ridges and valleys of similar dimensions as the pure hydrogel (Fig. 2C). Within
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the hydrogel, individual AgNW can be observed in the bulk hydrogel. As expected, the AgNWs are randomly distributed within the hydrogel. Randomly distributed AgNWs allow for the formation of conductive networks, as previously shown [34]. However, aligning AgNWs leads to a higher
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conductivity at the same nanowire concentration [35], which could allow reducing the material costs of the device. Additionally, X-ray spectroscopy was used to analyze the elemental composition of the
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composite material (Fig. 2D). Unsurprisingly, the two most abundant elements found were carbon and
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oxygen in nearly the ratio expected from the hydrogel. Additionally, almost 5wt% silver was found by elemental X-ray spectroscopy, which was higher than the expected value of 2wt%. As only a small
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sample, containing AgNW, was chosen for X-ray spectroscopy, the content of AgNW was not representative for the bulk material. Hence, the measured content of silver nanowires was higher than
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the expected value. Lastly, small amounts of sodium and chloride were found. These originated from salts in the hydrogel precursor in the synthesis of the hydrogel. Through the drying of the material prior to SEM analysis, the concentration was increased compared to the hydrated form of the
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hydrogels. We also characterized the mechanical properties of the PEG hydrogel/AgNW materials and their properties compared to other hydrogels (Fig. 3). Although one would expect an altered
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mechanical behavior of the hydrogel after the addition of AgNWs, the stress-strain curves for PEG and the PEG hydrogel/AgNW composite materials were similar and their compressive moduli were not significantly different. It is due to the amount of AgNWs (~0.2wt%). Although the desired electrical properties are achieved, The amount of AgNW (~0.2wt%) is too small to lead to a
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measureable difference in mechanical properties. As a result, we confirmed that the addition of low concentrations of AgNWs to PEG hydrogels did not significantly alter their mechanical properties. In contrast, the compressive modulus of the matrigel was significantly lower than that of PEG hydrogel/AgNW composite materials (Fig. 3B). Furthermore, we analyzed stress-strain curves for tension tests between PEG hydrogel/AgNW micropatterns on PET film and pure PET film (Fig. S1). As expected, it showed that they were almost identical. The addition of the supporting PET film can
lead to suitable mechanical properties. However, the PET film needs to be replaced by a biodegradable material to obtain a fully degradable neural implant or graft.
3.2 Simulation of electrical behavior Before conducting experiments, the electrical behavior of PEG hydrogel/AgNW micropatterns was simulated using Comsol Multiphysics software (Fig. 4). Firstly, the current density within the PEG
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hydrogel/AgNW micropatterns was simulated (Fig. 4). From the simulation, it can be seen that when applying a voltage the current density within the PEG hydrogel/AgNW structure is higher than the
current density of the surrounding medium and the supporting PET film. Simulations of the PEG
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hydrogel/AgNW microstructure parallel to the applied voltage have shown that the current density
within the structures would be less homogeneous. Hence, to enhance homogeneity, it was decided to use the PEG hydrogel/AgNW microstructure perpendicular to the applied voltage. The model further
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suggests that the simulated current density within the individual ridges of the PEG hydrogel/AgNW is inhomogeneous. This effect is related to the mesh size of the Comsol model and numerical
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instabilities during the simulation. In experiments, we expect a homogeneous current distribution
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within the individual ridges of the microstructure. Secondly, the average current at the wall in the different materials was simulated (Fig. 4C). It was observed that PET and PEG showed lower currents
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than AgNW due to the lower conductivity of the materials. For the PEG hydrogel/AgNW material, a current similar to AgNWs can be expected due to similar conductivities [33], showing the feasibility
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of the material in the conduction of electrical stimuli. Furthermore, the dependency of the current inside the ridges on the applied voltage was simulated (Fig. 4D). As expected from Ohm’s law, a liner
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relationship between current and applied voltage was found.
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3.3 NSC-derived neural differentiation To optimize the dimension of PEG hydrogel/AgNW micropatterns, we investigated various widths of PEG hydrogel/AgNW micropatterns without applying a voltage (Fig. 5). Choosing a width of the
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micropatterns smaller than the diameter of the neurospheres can lead to neurosphere growing on several microridges simultaneously. Through the spatial proximity of neurospheres on neighboring ridges, the fusion of several neurospheres was observed (Fig. 5A). If a PEG hydrogel/AgNW micropattern width wider than the diameter of the neurospheres is used, the guiding efficiency of neurites cultured in the micropatterns may be lost. This is observable by the radial outgrowth of neurites (Fig. 5C,D). Therefore, we used PEG hydrogel/AgNW micropatterns with 200µm width, which was similar to the diameter of neurospheres (Fig. 5B), showing the best compromise between
neurite guiding and neurosphere seeding efficiency. To determine the differentiation of NSCs into neurons in the PEG hydrogel/AgNW micropatterns, the neural markers (e.g., Tuj1, MAP2, and nestin) were assessed by specific immunostaining with fluorescently labeled antibodies (Fig. 6). From the fluorescent microscopy images, it was observed that a large number of cells differentiated into Tuj1postive neuronal cells. To gain further information about NSC differentiation behaviors, we used Map2 and nestin biomarker (Fig. S2). From the fluorescence microscopy images, we observed that a number of NSCs differentiated into MAP2-positive mature neuronal cells, while a small number of
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cells are nestin-positive neural progenitor cells. Furthermore, the outgrowth of neurites mediated from the electrical stimuli was observed. However, the strongest growth of neurites, both by number and length, was observed when applying intermediated voltages of 5 and 10V (Fig. 6 B,C,F,G). Longer
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neurites can be observed in the PEG hydrogel/AgNW micropatterns (Fig. 6G) compared to control experiments at the same voltage (Fig. 6C). Furthermore, while the direction of neurite growth appears to be random in control experiments, neurite outgrowth appears to follow the PEG hydrogel/AgNW
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micropatterns.
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3.4 Analysis of neurite outgrowth
The neurite outgrowth was measured with Image J and analyzed regarding neurite number and length
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(Fig. 7) and the growth direction (Fig. 8). In experiments conducted with the PEG hydrogel/AgNW micropattern, the average length of neurites was increasing with increasing applied voltages (Fig. 7A).
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Furthermore, the number of neurites increased with the applied voltage (Fig. 7B). Application of low voltages of 10V significantly increases the length of neurites compared to control experiments, while the number of neurites also increases compared to no voltage control experiments, confirming results
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by Patel et al. [36]. However, when applying a higher voltage of 20V, neither neurite length nor neurite number is significantly increased compared to control experiments without electrical stimuli.
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Similarly, Yamada et al. observed a lower differentiation efficiency at voltages of 20V compared to 15V [12]. The mechanism for this is still not fully understood [37]. A possible reason is that low electric fields that present in wound healing, affect the cell steering mechanism, while higher electric fields (>2V/cm) also stimulate overall cell motor activity [38]. In addition to the length and number of
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neurites, the direction of neurite outgrowth was also analyzed (Fig. 8). In control experiments, neurites could be observed growing in all directions, with a fairly even distribution (Fig. 8A). In literature a tendency of neurites to grow towards the cathode was observed [36]. However in our experiments this tendency was not observed. A possible reason for this might be that in our experiments the frequency of stimulation (f=139µHz) is four orders of magnitude lower than used in previous studies (f=1Hz) [36], which observed growth towards the cathode. In contrast, neurite growth orients along the ridges of the substrate when an electrical field is applied (Fig. 8B). In
experiments with no applied voltage, the direction of neurite outgrowth appears to be random, as observed in control experiments. Nevertheless, when a voltage of 5 or 10V is applied, the outgrowth of neurites orients itself along the ridges of the PEG hydrogel/AgNW micropattern. In direct comparison of the control experiment and the PEG hydrogel/AgNW micropattern at 10V, it can be seen that on the microstructure 60% of neurites follow the structure in their growth direction, while only 20% of neurites grow in the direction of the electrode for the control experiments (Fig. 8C). This superior performance of the PEG hydrogel/AgNW micropattern could be attributed to two different
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factors. Firstly, the ridges of the material impose spatial limitations to the growth of neurites, especially for neurites close to the edge of a ridge. This effect would be more visible with longer neurites, as observed in 10V experiments, as they are more likely to encounter an edge of the ridges.
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Secondly, AgNW in the composite material might aid as a guide to the outgrowth of neurites.
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4. Conclusions
We presented a conductive PEG hydrogel/AgNW composite micropattern-based sensor that was used
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for NSC differentiation into neurons under the application of electrical stimuli. Microstructures were
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formed from the composite material with the aid of a PDMS mold, through which neurite outgrowth could be guided. Simulation of the electrical behavior of the composite material and structure
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demonstrated the advantages of the PEG hydrogel/AgNW composite material compared to conventional hydrogels. Therefore, our PEG hydrogel/AgNW composite micropattern-based sensor
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NSC therapy applications.
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on a flexible, supporting PET film could allow the potential future use as a neural implant device for
Acknowledgements
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This research was supported by BioNano Health-Guard Research Center funded by the Ministry of
Science
and
ICT
(MSIT)
of
Korea
as
Global
Frontier
Project
(grant
number
H-
GUARD_2014M3A6B2060503), Republic of Korea. This work was supported by the National Foundation
(NRF)
of
Korea
grant
funded
by
the
MSIT
A
Research
(Grant
number
2016R1A6A1A03012845). This research was also supported by Basic Science Research Program through the NRF funded by the Ministry of Education (Grant number 2016R1A6A3A11931838). Authors appreciate Prof. Sun Woong in Korea University to kindly provide NSCs.
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Biographies
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Jong Min Lee received his B.S. degree in Biochemical Engineering from Korea Polytechnic University, Korea in 2010, respectively. In 2016, he received his M.S. and Ph.D. degree in Mechanical Engineering, both from Sogang University, Korea. Currently, he is a research professor for the Department of Mechanical Engineering at Sogang University, Seoul, Korea. His current research interests are development of microfluidic sensing devices for healthcare and medical applications. Joo Yoon Moon received his B.S. degree in Biomedical Engineering at Kyung Hee University, Korea in 2015 and received his M.S. from Sogang University, Korea (2017). His research interests are microfluidics and stem cells for diagnosis and treatment of disease.
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Tae Hyun Kim received the B.S. degree from Seoul National University of Science and Technology in Mechanical System design engineering (2012). He received his M.S degree in Mechanical Engineering in 2015 at Sogang University, Korea. Currently, he is a Ph.D. degree candidate at Department of Mechanical Engineering in Sogang University. His research interests are modeling and simulation of multi-physics in bio-medical application.
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Seung Won Lee received his B.S. degree at Hanyang University with Material Engineering in 2000. In 2017, he received his M.S. at Department of Mechanical Engineering in Sogang University, Korea. Currently, he is a Ph.D. degree candidate at Department of Mechanical Engineering in Sogang University. He is also Senior Manager for Platform Technology Lab, SAIT, in Samsung Electronics, Korea. His current research interests are fabricating sensing device for electrical applications.
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Christian D. Ahrberg obtained his Bachelor and Master Degree in Chemical and Bioengineering in 2012. He worked at Bayer Technology Services in Leverkusen before starting his Ph.D. under supervision of Andreas Manz at the Mechatronics department of Saarland University in cooperation with KIST Europe, during which he worked on a portable real-time PCR device. Since graduating in 2016, he is currently a postdoctoral researcher at the BioNano Technology Lab in Sogang University, Seoul, Korea.
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Bong Geun Chung received the B.S. and M.S. degree from Hanyang University, Seoul, Korea. In 2007, he obtained Ph.D. degree from Materials Science Engineering in University of California Irvine, USA. After Ph.D. degree, he worked as a Postdoctoral Research Fellow and an Instructor in Harvard Medical School, USA. Currently, he is an Associate Professor at Department of Mechanical Engineering in Sogang University, Seoul, Korea. His main research interests are development of functional 3D Lab-on-a-Chips, Biosensors, and Nanomaterials to direct the stem cell and cancer fate.
Figure Legends Fig. 1. Schematic of the production of PEG hydrogel/AgNW micropattern-based sensor (A). Photograph of the PEG hydrogel/AgNW micropattern inside of the PDMS mold. PEG hydrogel/AgNW micropattern is stained with red dye. Scale bar is 1cm (B). Fluorescent microscopy image of PEG hydrogel/AgNW micropattern after removal of the PDMS mold. For illustration
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purposes the hydrogel is stained with fluorescein dye. Scale bar is 100µm (C).
Fig. 2. SEM images of the PEG hydrogel (A), AgNW (B), and PEG hydrogel/AgNW composite
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material (C). X-ray element spectroscopy of composite PEG hydrogel/AgNW composite material (D).
Fig. 3. Stress-strain curve of compression test of different hydrogel materials (A) and analysis of compressive modulus (B). The compressive modulus is significantly different between PEG
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hydrogel/AgNW composite material and matrigel (**p<0.01).
Fig. 4. Simulations of electrical behavior of the composite PEG hydrogel/AgNW micropattern.
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Schematic of experimental setup for applying electrical stimuli (A). Simulated current density in the micropattern with an applied voltage of 10V (B). Analysis of simulated average current for different
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materials at an applied voltage of 10V (C) and current for the PEG hydrogel/AgNW composite
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material at different applied voltages (D).
Fig. 5. Fluorescent microscopy images of NSCs on PEG hydrogel/AgNW micropatterns with 100µm
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(A), 200µm (B), 300µm (C), and 500µm (D) width. A green dye was used for Tuj1, red for nestin, and blue for DAPI.
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Fig. 6. Fluorescent microscopy images of NSCs after culture in control experiments (A-D) and PEG hydrogel/AgNW micropatterns (E-H). To facilitate NSC differentiation, various electric stimuli are applied. Cells are immunostained by TuJ1 (green) and cell nuclei are stained by DAPI (blue). Scale bars are 200µm.
Fig. 7. Analysis of average neurite length (A), average number of neurites (B) for NSCs cultured on the PEG hydrogel/AgNW micropattern.
Fig. 8. Rose diagrams indicating percentage of neurites growing in a certain direction in control experiments (A) and in experiment conducted on the PEG hydrogel/AgNW micropatterns (B). Rose diagram comparing the percentage of neurites growing in a certain direction of control and
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micropattern experiments (C). 0˚ is growth towards the top of the micropattern and 90˚ is growth towards the cathode, respectively. Growth was classified into eight different directions, each
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