Biomaterials 53 (2015) 358e369

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Electrical stimulation by enzymatic biofuel cell to promote proliferation, migration and differentiation of muscle precursor cells Jae Ho Lee a, b, Won-Yong Jeon a, c, Hyug-Han Kim c, Eun-Jung Lee a, b, Hae-Won Kim a, b, d, * a

Department of Nanobiomedical Sciences and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 330-713, Republic of Korea b Institute of Tissue Regeneration Engineering, Dankook University, Cheonan 330-714, Republic of Korea c Department of Chemistry, College of Natural Science, Dankook University, Cheonan 330-714, Republic of Korea d Department of Biomaterials Science, College of Dentistry, Dankook University, Cheonan 330-714, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 December 2014 Received in revised form 11 February 2015 Accepted 13 February 2015 Available online

Electrical stimulation is a very important biophysical cue for skeletal muscle maintenance and myotube formation. The absence of electrical signals from motor neurons causes denervated muscles to atrophy. Herein, we investigate for the first time the utility of an enzymatic biofuel cell (EBFC) as a promising means for mimicking native electrical stimulation. EBFC was set up using two different enzymes: one was glucose oxidase (GOX) used for the generation of anodic current followed by the oxidation of glucose; the other was Bilirubin oxidase (BOD) for the generation of cathodic current followed by the reduction of oxygen. We studied the behaviors of muscle precursor cells (MPCs) in terms of proliferation, migration and differentiation under different electrical conditions. The EBFC electrical stimulations significantly increased cell proliferation and migration. Furthermore, the electrical stimulations promoted the differentiation of cells into myotube formation based on expressions at the gene and protein levels. The EBFC set up, with its free forms adjustable to any implant design, was subsequently applied to the nanofiber scaffolding system. The MPCs were demonstrated to be stimulated in a similar manner as the 2D culture conditions, suggesting potential applications of the EBFC system for muscle repair and regeneration. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Electrical stimulation Enzymatic biofuel cell Muscle precursor cells Implantable device Muscle regeneration

1. Introduction The physiology of skeletal muscle is mediated by two major factors: one is Electrical stimulation from nerve cells and the other is mechanical stimulation [1,2]. Electrical stimulation from motor neurons is known to be critical in regulating the development and maintenance of myoblasts, such as proliferation, migration and differentiation for myotube formation [3e6]. The skeletal muscle atrophy characteristic of the common motor neuron disease, amyotrophic lateral sclerosis (ALS), is caused by the lack of stimulation from motor neurons [7]. Most ALS patients die within 5e6 years after diagnosis [7,8]. Nevertheless, proper clinical therapy does not yet exist to relieve the symptoms of ALS [7].

* Corresponding author. Institute of Tissue Regeneration Engineering, Dankook University, Cheonan 330-714, Republic of Korea. Tel.: þ82 41 550 3081; fax: þ82 41 550 3085. E-mail address: [email protected] (H.-W. Kim). http://dx.doi.org/10.1016/j.biomaterials.2015.02.062 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

To this end, several strategies to mimic electrical stimulation by neurons have been explored, including the use of electrical biophysical systems and electrical implantable biomaterials [9e12]. Histological electrical therapies such as pain management, improving neuromuscular functioning, and allowing joint motility and tissue repair are now clinically available [13e17]. Direct current is also used clinically for electrical stimulation [18,19]. However, the effective mechanisms governing those therapies have yet to be clarified [20e22]. More extensive studies in vitro have been piled up to investigate the electrical stimulatory effects and to elucidate the underlying mechanisms at the cellular level [1,6,14,23,24]. Cumulative findings have revealed that the exogenous electrical stimuli provided by electrotherapeutic devices significantly influence cell structure, movement, metabolism, replication, proliferation, and differentiation [14,16], which is reasoned to be primarily due to the stimulation of cell membrane receptors and alterations in membrane ion channel characteristics [25]. Systems for providing electrical stimuli have been possible through electrical circuits using electrical power sources mostly

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made of inorganic materials [17,26]. However, these have limited life time, low biocompatibility and often require a sizable device, restricting extensive use and ease of clinical applications. This is particularly limiting when designing electrical stimulation systems for implantable biomaterials. Implantable biomaterials in diseased and dysfunctional muscle tissues should be biocompatible (and possibly degradable) with sizes and shapes that allow for implantation as well as ease of repair and regeneration of associated tissues. Biomaterials (i.e., degradable biopolymers) generally available for implantable purposes meet those basic criteria. However, intrinsic formation of electrical circuits in the currently available biomaterials is impossible. Here we present enzymatic biofuel cell (EBFC) as an electrical power source that can intrinsically generate electrical stimuli for muscle repair and regeneration. EBFC has recently been recognized as a promising power source for implantable biomedical devices in the living body [27]. In 1911, Potter introduced the first biofuel cell using cultured yeast and E. Coli cells on platinum electrodes [26]. During the 1960s in the United States, practical interest has been developed in the use of EBFC as an efficient energy production system for vehicle power suppliers and as an implantable power source for cardiac pacemakers [28]. However, the electrochemical performance in all of the early EBFC studies was very poor. Efforts have recently been devoted to improving EBFC and overcoming limitations such as the short active lifetime, low power density, and low efficiency for electrical devices [29e32]. With improvement, the applicability of EBFC to other kinds of designable formulations has great merit for implantable uses in tissue repair and regenerative therapy. Another important advantage of EBFC is good biocompatibility because the glucose/oxygen that exists at high concentrations in human body plasma can serve as fuel for EBFC. Despite these fascinating features of EBFC, no studies have yet been conducted using EBFC as an electrical stimulation system for the repair and regeneration of tissues including muscle. Here we report the utility of EBFC for applications in tissue repair and regeneration targeting muscle tissue. Depending on the type of enzymes used, EBFC can be categorized into anodic, cathodic and fullset conditions, consequently providing electron-rich and electronpoor conditions to the cell and tissue microenvironment. In the cathodic compartment, Bilirubin oxidase (BOD) consumes electrons by reducing dioxygen (O2) to water (H2O), resulting in an electronpoor condition in the vicinity of the cathode [33,34]. In the anodic compartment, glucose oxidase (GOX) releases electrons by oxidizing glucose to gluconolactone, creating an electron-rich condition [33,34]. EBFC can therefore easily create two completely different electrical environments for the cell and tissue. The gradient electrical microenvironment is essential for action potentials in muscle cells. Using EBFC-derived electron-rich or -poor conditions, we investigated the effects on the behaviors of the muscle precursor cell C2C12, such as cell proliferation, migration and differentiation. The results provided here will be highly useful for providing evidence on utilizing EBFC for the repair and regeneration of muscle as well as for possible extended utility in other tissues. 2. Materials and methods 2.1. C2C12 cells Cells were purchased from American tissue culture collection (ATCC). Cells were thawed and plated at a density of 2  103 cells/cm2 in culture dishes using Dulbecco's modified eagle medium (DMEM; LM 001-05, WelGENE, Daegu Korea) supplemented with 10% fetal bovine serum (FBS; Hyclone, Thermo), and 1% penicillin/ streptomycin (Giboco) at 37  C in a humidified atmosphere containing 5% CO2. 2.2. Fabrication of EBFC Screen-printed carbon electrodes (SPCEs) were prepared with Electrodag® 423SS (Acheson, Port Hurton, USA) on OHP film using a semi-automatic screen printing machine. The anodic enzyme was glucose oxidase (GOX) from Aspergillus

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niger (219 U/mg) purchased from Amano Enzyme Inc. (Japan). Cathodic catalyst was Bilirubin oxidase (BOD) from Myrothecium verrucaria (10.5 U/mg, Sigma enzyme). Poly (ethylene glycol) (400) diglycidyl ether (PEGDGE) supplied by Polysciences, Inc was used as a cross-linker. The loading solutions for anodic and cathodic electrodes were composed of enzyme, redox mediator, and cross-linker. The anodic catalyst consisted of the cross-linked adduct of 40 mg/ml of GOX, 2.0 mg/mL of PVI-[Os(4,40 dimethoxy-2,20 -bipyridine)2Cl]þ/2þ, and 40.0 mg/mL of PEGDGE with the volume ratio (4: 4: 1). The cathodic catalyst consisted of the cross-linked adduct of 40 mg/ml of BOD, 2.0 mg/mL of PVI-[Os(4,40 -dimethoxy-2,20 -bipyridine)2Cl]þ/2þ, and 40.0 mg/ mL of PEGDGE with the volume ratio (4: 4: 1). 10 ml of the mixture was placed on the SPCE, and dried for 24 h in the desiccator at room temperature (25 ± 1  C). 2.3. Electrochemical characterization of EBFC For electrochemical measurements, two SPCEs were glued onto the edge of the dish (35 pie dish). The anodic or cathodic electrodes used in this study were 15-mmlong and 2-mm-width, with active areas of 30 mm2. A two-electrode electrochemical cell coupled to a CHI 660B potentiostat (Austin, TX, USA) was used for the open circuit potential (OCP) and i-t technique. The electrochemical characteristics of modified PVI-[Os(dmo-bpy)2Cl]þ/2þ were studied with 3.5 mm-diameter working electrodes (SPCEs) on a flexible polyester film. A 0.5-mm-diameter platinum wire counter electrode and an Ag/AgCl micro-reference electrode (3.0 M KCl saturated with AgCl, Cypress, Lawrence, KS, USA) were used. The electrodes were placed on the culture dish with medium and cells similar to the cell culture conditions (detailed in the following sections), and the current density was monitored with respect to exposure time. The power density and polarization curves were obtained by a cyclic voltammetry technique [35]. The applied potential ranged from 0.4e to 0.6 V and the scan rate was 1 mV/s. The experiment was carried out under ambient air conditions at room temperature. 2.4. EBFC set-up for 2D cell cultures C2C12 cells were seeded at 1  105/ml on the culture dish (35 pie dish) set up with three different EBFC designs or without EBFC. A 3 mm gap was left between the EBFC electrode and bottom of the culture dish. 2 ml of culture medium was added initially to level the medium below the electrodes, which is required for the cells to initially settle down and anchor to the culture dish. After 16 h, 3 ml of culture medium was added to completely cover the electrodes for enzymatic reaction. From this time point (t ¼ 0), the cells were cultured for predetermined time points for required assays. 2.5. Live imaging of cells A live image of cells was monitored to visualize the cell motility and adhesive protein arrangement. For this, cells were co-transfected with red fluorescent protein (RFP)-paxillin and green fluorescent protein (GFP)-actin. RFP-paxillin plasmid was kindly provided by Dr. Xiong from Georgia Health Science University. GFP-actin plasmid was purchased from BD pharmingen™ (Green FP vector-actin: 558721). Neon transfection system (Invitrogen life science) was used for the cell transfection under optimized electroporation condition (1100 V, 30 ms, and 1 pulse). After 3 days of culture, paxillin-RFP/actin-GFP co-transfected cells were trypsinized and prepared into a cell suspension (1  104/ml). The suspended cells were seeded on the confocal dish (100350, SPL Korea) for live imaging. Red and green positive cells were selected and the live image processed every 2.5 min for 20 cycles, by confocal laser scanning microscopy (META M700 ZEISS, Germany) for a total 50 min. 2.6. Cell count After culturing the cells for different time points, cells were harvested by trypsinization and the cell number was determined by trypan blue exclusion and hemocytometer counting. 2.7. Cell migration study; wound closure model For the in vitro cell migration study, the cell suspension was prepared at 5  105/ ml in culture medium. Cell suspension of 75 ml was applied to each culture well. After culture for 24 h to attain a near cell-confluence, a cell-free gap was made using the culture-insert tool (80206, ibiTreat, ibidi, Germany), which creates a cell-free gap with a width of ~500 ± 50 mm. The medium was refreshed gently to eliminate any non-adherent cells. After 16 and 24 h of culture, the number of cells found within the gap was counted. 2.8. Adhesion protein rearrangement and cell polarization behaviors To visualize the morphology of the cells, cultured cells were fixed with 4% paraformaldehyde. Next, the samples were subjected to regular immunocytochemistry procedures; treated with rabbit anti-focal adhesion kinase (FAK) antibody (A-17; 1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4oC then with the anti-rabbit secondary FITC-conjugated antibody, phalloidin (A34055, Alexa Fluor 555 phalloidin, Invitrogen) for F-actin staining at room temperature for 1 h. The nuclei were counter-stained with DAPI. After washing with PBS, the sample was mounted by anti-faded medium (H-1000, Vectashield mounting media, Vector

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labs). A Zeiss M700 CLSM was used for image processing. The localization of FAK and F-actin patterns in the cells were observed. The polarization of cells was analyzed using confocal images. For this process, confocal images of cells in a wide area were taken. Specifically, a total area of 40  8 mm was imaged starting from the electrode zone, and each distance frame (5  8 mm) from the electrode was considered for cell polarization analysis. The position of the nucleus, the total cell area and the center of the cell area was noted for each cell. Using this data, the displacement of the nucleus from the center of the cell area was indexed. When the center of the cell area is at a different location than the nucleus, the cell is considered to be polarized, and the polarized cell percentage in each distance frame was recorded.

fluid jet was ejected. To orient the nanofibers in one direction, a cylindrical metal collector (15 cm distanced from the needle tip of the needle) rotating at a speed of 900 rpm was used. The electrospun nanofiber was completely dried, washed three times in ethanol, and dried for further cellular study. The surface morphology of the electrospun nanofibers were examined using a high resolution scanning electron microscope (SEM; JEOL-JSM 6510). The average diameter of nanofibers was calculated from the SEM images obtained at random locations. For EBFC loading, the enzymes at low concentration (40 mg/ml) were simply pasted onto the nanofiber scaffolds.

2.9. Myogenesis observation by RT-PCR gene expressions

The analyses of C2C12 cells cultured on the nanofiber scaffolds were performed similar to the 2D cultures, which include cell proliferation (cell count), morphology (confocal images), and myogenic differentiation (RT-PCR gene expression, immunocytochemistry, and Western blotting).

For the myogenic differentiation of C2C12 cells, the regular culture medium was replaced by the differentiation medium consisting of a mixture of AIM and serumfree DMEM at a ratio of 2:1. To investigate the myogenesis of cells, the expression of genes was first analyzed by RT-PCR. Genes related with myogenesis, including MyoD, myogenin, and a-actinin, were considered, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the reference gene. The primer sequence of each gene is as follows: For MyoD, (F) 5-GGAGTGGCAGAAAGTTAAG-3 and (R) 5ACGGGTCATCATAGAAGTC-3; for myogenin, (F) 5-GGATATGTCTGTTGCCTTC-3, and (R) 5-TGGGTGTTAGCCTTATGT-3; for a-actinin, (F) 5-GGACTACACTGCCTTCTC-3, and (R) 5-CAGCCTATACTTCAGCCTTTA-3; and for GAPDH, 5-GAAACCTGCCAAGTATGATG3, and (R) 5-GGAGTTGCTGTTGAAGTC-3. Total RNA was isolated from each cell pellet using an RNA isolation kit (RNeasy mini kit 74104, Qiagen). RNA samples (1 mg) were reverse transcribed to cDNA in 40 ml reactions using the Quantitect RT kit (#205311, Qiagen) according to manufacturer's protocol. The reaction was allowed to proceed at 95  C for 5 min. A similar reaction mixture without the reverse transcriptase enzyme was prepared and used as a template to demonstrate the absence of contaminating genomic DNA. One microliter of cDNA was subjected to PCR amplification using each specific gene primers with a pre-mixed PCR kit (Bioneer, Korea). PCR reactions were conducted using 35 cycles at 95  C for 30 s, 60  C for 30 s, then 75  C for 60 s and were performed in triplicate for each cDNA. The PCR products were run on 2% agarose gel for 25 min at 100 V. The sample band intensity was quantified using the software program Gene tools ver 4.01 (Syngene UK), and normalized to that of endogenous GAPDH. 2.10. Myogenesis observation by immunocytochemistry The co-fluorescence immunocytochemistry was targeted for specific myogenesis markers including myogenin and a-actinin. Cells were fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100 for 10 min. The samples were then blocked with 10% goat serum for 30 min, after which they were probed with primary antibodies overnight at 4  C. The samples were washed three times in PBS, and fluorescence image was developed by incubation with donkey anti-rabbit secondary antibodies conjugated with Alexa fluor 555 (A31572, Alexa Fluor, 1:5000, invitrogen biotechnology) for anti-a-actinin antibody and goat anti-mouse secondary antibody conjugated with FITC (SC2012, 1:500, Santa Cruz biotechnology) for anti-myogenin antibody for 1 h at room temperature. The nuclei were co-stained with DAPI. Stained cell images were processed under a confocal microscope (M700 ZESS, Germany). 2.11. Myogenesis observation by western blot protein analysis The protein levels of cells were analyzed by Western blot with antibodies against myogenin (5FD; SC52903, 1:1,000, Santa Cruz Biotechnolgy US), and a-actinin (SC15335, 1:1,000, Santa Cruz Biotechnology, US). The cells were harvested and treated with commercial lyses buffer (iNtron Biotechnology). Sample solution was then heated with 20 ml 5 loading buffer for 5 min, which was then loaded to each well of an 8% SDS/PAGE gel and trans-blotted to the nitrocellulose membrane for 150 min at 350 mA. The trans-blotted membrane was blocked by 5% fat-free milk Tris buffer with 0.5% Tween-20 for 1 h at room temperature. The primary antibody was diluted in 2% fat-free milk buffer solution and incubated overnight in a cooled chamber with the transferred membrane. The blotted membrane was washed by 0.5% Tris buffer and incubated with horseradish peroxidase-conjugated anti-mouse secondary IgG, and immunoreactive bands were detected using ECL detection reagent (Western Bright ECL, K-12045, Advansta Cop). The sample band intensity was quantified using the software program Gene tools ver 4.01 (Syngene UK), and normalized to that of endogenous b-actin. 2.12. Nanofiber preparation and EBFC loading For the preparation of nanofiber scaffolds, biopolymer composites made of polycaprolactone and gelatin were prepared using an electrospinning technique. Polycaprolactone (Mw 80,000), gelatin (type B, bovine skin) and tetrafloroethanol (TFE) were all obtained from SigmaeAldrich. Both polycaprolactone and gelatin prepared at a concentration of 15 wt% in THF were homogenized at 1:1 while vigorously stirring for 2 h. Five milliliters of the solution was electrospun using a 10 ml syringe with a needle diameter of 0.4 mm, at a flow rate of 1 ml/h. A high voltage (11.5 kV) was applied to the tip of the needle attached to the syringe while a

2.13. Cellular assays on nanofiber scaffolds

2.14. Statistical analysis Experiments were performed in triplicate, unless otherwise specified. Data are expressed as mean ± one standard deviation. Statistical analysis was carried out using one way analysis of variance (ANOVA) followed by a Fisher post-hoc test and a significance level was considered at p < 0.05.

3. Results and discussion 3.1. Anode/cathode performance in EBFC A simple EBFC design to generate electric signals either at the anode or cathode is depicted in Fig. 1a. Depending on the enzyme used, EBFC can be divided into anodic, cathodic, or anodic/cathodic conditions. The GOX enzyme releases electrons by oxidizing glucose to gluconolactone, resulting in an electron-rich condition at the anode, while the BOD enzyme consumes electrons by reducing oxygen to water, creating electron-poor culture conditions at the cathode. Accordingly, a full-set EBFC can be set up by loading both GOX and BOD a certain distance apart. The resultant electron-rich and electron-poor conditions at each electrode generate a flow of electrons (current flow in the opposite direction) (Fig. 1b), which will affect the cells present. The current density generated by EBFC was measured to characterize the electrochemical performance of the system. The enzyme concentration was varied and the redox mediators were accordingly adjusted as described in the ‘Materials and methods’ section. The redox mediators used in this study can overcome the poor electron transfer between enzymes and electrodes as they are readily diffusible and satisfactorily shuttle electrons between the biocatalysts and the electrodes. Some representative conditions were first plotted (Fig. 1c) and showed that the current density was well stabilized over the time course measured. Furthermore, the results showed that GOX loaded at a low concentration of 40 mg generated low negative current density (1.878 ± 0.35 nA/cm2) while BOD loaded at an equal concentration (40 mg) created low positive current (8.676 ± 1.17 nA/cm2). The full-set system with GOX/BOD loaded at 40 mg had a positive current density (14.07 ± 1.63 nA/cm2). The current density could be controlled by modifying the enzyme loading concentration; increasing the enzyme concentration (from 40 to 1000 mg) increased the current density gradually, as evidenced by a full-set condition (Fig. 1d). Furthermore, the powder density and open circuit potential of the EBFC could be obtained (Fig. 1f), based on the cyclic voltammograms of the anode and cathode electrodes (Fig. 1e) when operated with using different enzyme concentrations (40e1000 mg/ml). The EBFC exhibited open circuit potential values between 0.503 V and 0.591 V, and provided maximum powder densities of 1.19, 2.41, 3.61 and 3.60 mW/cm2 at 40, 200, 500 and 1000 mg/ml, respectively. These values of power densities were much lower than those reported elsewhere [31], which might allow the current EBFC system to be applied for the culture of tissue cells.

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Fig. 1. (a,b) Schematic showing the EBFC-generated redox potential in culture system (a) and the electron transfer around the cells in culture conditions (b). (c) Current density recorded with time under different EBFC conditions (low enzyme concentration representatively shown), and (d) the variation of current density with difference in enzyme concentration (from 40 to 1000 mg/ml) for full-set condition representatively shown. (e) Cyclic voltammograms (current density vs. potential) of anode and cathode electrodes recorded with using different concentrations of enzyme (from 40 to 1000 mg/ml), and (f) open circuit potential and power density curves presented as a function of current density that obtained based on the cyclic voltammetric results (open symbols: circuit potential, and closed symbols: power density).

For the culture of C2C12 cells, we first sought EBFC conditions that are favorable for cell survival by adjusting the concentrations of the enzyme and the redox mediator. We compared cell survival under two opposite conditions where the concentrations of both enzyme and redox mediators differed dramatically. At low amount of enzyme (40 mg), the limiting currents for anodic, cathodic, and full set EBFC conditions were in the range of a few to tens of nA/cm2. However, at extremely high amount (10 mg), the limiting current densities increased substantially; 495.2 ± 24.3 nA/cm2,

2228.9 ± 94.2 nA/cm2 and 10,867 ± 549 nA/cm2, respectively for anodic, cathodic, and full set EBFC. Under these different electrical current levels, the cell morphology was visualized using confocal microscopy after immunofluorescence staining for nuclei (blue), FAK (green) and Factin (red) at 6 h of culture, as shown in Fig. 2. In particular, the FAK immunocytochemical stain showed the cytoskeletal distribution of adhesive molecules at the early stage of the cell event. The cells without EBFC-loading showed active cytoskeletal processes with

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many FAK-signaled focal contacts at the cell periphery. The cells cultured under low enzyme loading conditions were also highly viable with active cytoskeletal extensions, clear FAK signals and sound nucleus formations. A closer examination of FAK signals, however, showed a clear difference between the groups. Although FAK signals were typically found at the periphery of cells with strong focal contact points when no EBFC was applied, the FAK signals were mostly observable within cytoskeletal regions and even around the nucleus in a highly diffused manner without a typical focal contact formation at the cell boundary. This FAK signaling was also observed in the cells when the enzyme loading was high. With high enzyme loads however, the cells showed significant morphological changes including the loss of general focal adhesion patterns, fragmented cytoskeletal network formation and closeting patterns at the nuclear area. We further analyzed the initial response of C2C12 cells under the EBFC electrical stimuli, by live-monitoring the cells for 1 h after transfection with red fluorescent protein (RFP) for paxillin molecules and green fluorescent protein (GFP) for F-actins (representative live images shown in Fig. S1). Compared to the cells cultured without EBFC stimuli, those with low electrical signal showed active actin fiber rearrangements with profound spreading over time as well as significantly higher RFP/GFP signal intensities. On the other hand, cells with high electrical stimulus showed a sort of cytoskeletal contraction with limited extensions. Based on these results, it is deduced that low electrical stimulation favors more dynamic redistribution of focal adhesion molecules including FAK and paxillin in cells, and this phenomenon hints at possible subsequent cellular processes that are influenced, including actin fiber rearrangement, cellular motility, proliferation and differentiation [36e40]. In particular, the cytoskeletal redistribution of adhesive molecules shifting from focal contact points have recently been highlighted to illustrate the different roles of the molecules in cellular processes, such as differentiation, from the typical initial anchorage events [41]. For example, the FAK signals were rearranged from focal points to internal cytoskeletal components in diffused form in C2C12 cells, and this change has been implicated as an indication of myoblastic differentiation [41e43]. The low enzyme concentration that generates electrical stimuli appropriate for cell survival was therefore used in the following experiments. 3.2. Effects on cell proliferation, polarity and migration by EBFC electrical stimulation The effects of EBFC electrical stimuli on cell behaviors were quantitatively examined by measuring the proliferative potential over a 24 h period. Cells initially seeded at 1  105 increased slightly at 16 h with a similar proliferation rate for all the EBFC conditions.

However, by the end of the 24 h period, cell proliferation increased substantially. The increase was more significant with EBFC electrical stimulations while there was no difference between the modes of electrical stimulation (Fig. 3a). In fact, previous studies have reported the improvement of cell proliferative potential by electrical signals introduced by external circuits other than EBFC [14,16]. We next observed the cell morphology under confocal microscopy after co-staining for nuclei (blue), FAK (green) and F-actin (red). We observed that the cell shape and nuclei disposition (polarity) were variable depending on the position of the cells (mainly dictated by the distance from the electrode). As shown in Fig. 3b, characteristic morphologies of cells positioned either in close proximity to the electrode (at ‘0’) or far from the electrode (distanced ‘2d’ from the electrode) were examined. Compared to those without EBFC, the cells with electrical stimulations generally had a higher population. In addition, cells at a distance from the electrode (at ‘2d’) retained normal morphology without exhibiting cellular polarity, however, the cells in close proximity to the electrode (at ‘0’) were highly polarized with clear nuclei dispositions and a gathering of FAK signals at the migration fronts, a phenomenon broadly observed in all EBFC modes. To further examine the distance-dependent cellular polarization influenced by the electrical stimuli, we imaged the cells cultured under different conditions within a distance of 40 mm (Fig. 3c). The shape of both polarized and unpolarized cells was analyzed at specific distance regions (every 5 mm up to a total of 40 mm). For convenience, cells with a nucleus displaced from the cell's center of mass were considered polarized. In normal cells without electrical stimuli, approximately 5e10% of cells were polarized despite their location. Cells with EBFC-generated electrical signals displayed significantly altered cellular polarization. Cells in close proximity to the electrode were ~22e27% polarized while ~15% of the cells at a distance of 40 mm were polarized. Though the cell polarization was largely distance-dependent in anode and cathode sets, i.e., gradually decreased with increasing distance from electrode, the polarization in full-set condition had more fluctuations with distance. It is therefore clear that the electron surplus or consumption condition is more attractive for cellular polarization. Since the polarized cells generally have high potential for migration [44,45], we next investigated the effects of EBFC electrical stimuli on cell migration. An in vitro wound closure model made of a 500 mm gap formed in the cell layer was constructed (as depicted in Fig. 4a), and the cell population in the gap during culture was measured at varying time points under different electrical stimuli. Optical cell images showed that a substantial number of cells migrated toward the gap at 16 h in the electrically stimulated groups, a behavior that was not readily apparent in the absence of

Fig. 2. Effects of current density generated by EBFC on cell survival and adhesive molecule distribution. Two inverse current densities (14.072 and 10867 nA/cm2) were generated using low (40 mg) and high (10 mg) loading of GOX/BOD enzymes. Cells without EBFC loading (‘w/o’) were tested as the control group. Cell spreading morphology and viability affected by the electrical stimulus were visualized by immunofluorescence micrographs after co-staining with nucleus (blue), F-actin (red), and FAK (green). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. (a) Cell viability with different EBFC culture conditions (anode; cathode; or full-set) at 16 and 24 h of culture. (b) Some representative confocal images showing different cell shapes (particularly polarization) at difference distances from the anode or cathode electrode (at the proximity ‘0’ and far distanced ‘2d’). (c) Full screening of the cells to reveal cellular polarization depending on the distance from the electrode and the EBFC signaling mode, and (d) the quantification of cellular polarization ratio. (Statistically significant difference noticed for n ¼ 3, *; P < 0.05 vs. control, by ANOVA with Fisher post-hoc test).

electrical stimuli. At 24 h, the in vitro gap was almost completely filled with cells in the electrically stimulated groups (Fig. 4b). The quantified number of cells demonstrated significant improvement in cell migrations toward the gap, and interestingly this phenomenon was most significant in the electron poor group (cathode) but was similar in the anode and full-set conditions (Fig. 4c). In fact, endogenous electrical stimulation in the wound healing process is known to be an important initial step for the repair of injury [6,16]. Several studies have also reported that external-circuited electrical stimulation provided directional cell migration cues and also mediated cell speed [6,46]. Thus far, the EBFC-generated electrical signals at mild levels were demonstrated to significantly contribute to C2C12 cellular proliferation, polarization and migration. 3.3. Effects on myogenic differentiation of cells by EBFC electrical stimulation We next investigated the effects of EBFC electrical stimulation on myogenic differentiation in C2C12 cells. First, the expression of mRNA levels associated with myoblasts, including myogenin,

myoD, and a-actinin, was analyzed using RT-PCR (Fig. 5a). GAPDH was used as the control gene. Without EBFC, there was little expression signal observed for all genes except a-actinin which indicated only a glimpse band intensity. On the contrary, the expressions of all genes were clear and substantial in all the EBFCstimulated conditions. Particularly, the cathode and full-set conditions appeared to reveal slightly stronger band intensities of genes compared to the anode condition (as also noticed in quantified band intensities). After confirming the gene level, the protein expressions were assessed by immunocytochemical staining of cells with characteristic markers at relatively late stages (as visualized in Fig. 5b). Myogenin and a-actinin proteins were both highly expressed in all the EBFC-conditions. Furthermore, Western blot analyses were used to determine the amount of protein expressions in the cells (Fig. 5c). Substantially higher expressions of both proteins were observed in the electrically stimulated groups. It is known that (pre)myoblasts secrete stage specific transcription factors during myogenesis and also substantially alter their morphological features [2]. From the early to late stages of myogenesis, the secretion of markers progresses from Myof and

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Fig. 4. Cell migration study using in vitro wound closure model. (a) Sketch of the model under different EBFC modes, (b) optical images of cells migrating towards the wound gap at different culture periods, and (c) quantified cell number migrated toward the gap. (Statistically significant difference noticed for n ¼ 3, *; P < 0.05 vs. control (w/o), and a,b; P < 0.05 between EBFC groups at each culture period, by ANOVA with Fisher post-hoc test).

MyoD, followed by myogenin and later to a-actinin and skeletal actinin [47]. At the same time, myogenic differentiation and maturation defines the sequential time frame of morphological changes including differentiation to myoblasts, fusion of myocytes, and myotubular formation [47]. Therefore, the current evidence on the series of myogenic markers indicates that EBFC-conditioned electrical stimulation drove the muscle precursor cells to develop well into a myogenic differentiation scenario, expressing a set of genes and proteins involved in the early to late myogenesis process. 3.4. Proof-of-study for the utility of EBFC using nanofibrous scaffolds After confirming the significant stimulatory effects that EBFC electrical stimulation have on C2C12 cellular proliferation,

migration and myogenic differentiation, we next sought to utilize the EBFC system as an implantable device for the purpose of muscle repair and regeneration. For this, a model scaffolding template, which is considered useful for populating cells and possibly for allowing muscle regeneration, was introduced. Well aligned nanofiber scaffolds made of blended biopolymer (PCL-gelatin) were fabricated to provide nanotopological pseudo-3D matrix cues to the cells, and EBFC was loaded onto the matrix in anode, cathode or full set conditions. SEM images exhibited nanofibrous matrices aligned (or randomized for a comparison group) with fiber sizes of approximately 930 nm (±264 nm) (Fig. 6a). On the nanofiber scaffolds, the EBFC designs were set-up equally in the culture dish, as the enzymes can be easily pasted by coimmobilization with mediators as described in the ‘Materials and methods' section. The C2C12 cells were then loaded and cultured

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Fig. 5. Myogenic differentiation of C2C12 cells affected by the EBFC-driven electrical stimulation. (a) RT-PCR analysis of genes related with myogenesis of C2C12 cells, including myogenin, myoD, and a-actinin. GAPDH used as a reference gene for measuring band intensities. Substantial expressions of all genes in the cells noticed when cultured under the EBFC conditions. (b) Immunofluorescence images showing the expressions of representative myogenic proteins (a-actinin and myogenin), and (c) Western blot analysis revealing protein expressions with significantly stronger intensities in the EBFC cultures. Band intensities quantified when normalized to b-actin.

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under EBFC-driven electrical stimulation. The cell proliferation measurements showed significant improvement with electrical stimulations particularly after 3 days of culture (Fig. 6b). The stimulation was similar to that previously described for the nanofiber-absent 2D culture dish; however, the stimulation time point appeared to be slightly retarded e this might be due to the fact that the initial cellular anchorage and stabilization on the nanofiber scaffolds were slower than on the culture dish. However, as soon as the cells were stabilized on the substrates, they could effectively sense the electrical stimuli. Next, the cellular morphology was visualized under confocal microscopy (Fig. 6c). On random nanofibers, a higher number of cells were observed in the electrically-stimulated groups than in the groups without stimulation. On the aligned nanofiber matrices, cells appeared to align even without electrical stimulation, and with electrical stimulation

in particular, the aligned cells formed some end-to-end contact points in a row which is different from the case in random nanofibers. It is therefore considered that the underlying topological cues might have effects on cellular alignment during differentiation. In fact, it has been shown that the aligned nano- or micropatterned surfaces were effective in lining up muscle precursor cells and in driving their differentiation into myoblast and myotubular formation, signifying the importance of the underlying topological alignment cues [1]. Thus the current findings address both the topological cues and the electrical signals. We further investigated the effects of the EBFC-integrated nanofibrous scaffolds on the myogenic differentiation of cells at the gene and protein levels. The RT-PCR gene analyses clearly revealed much stronger expressions of MyoD, myogenin, and aactinin genes in the cells cultured under electrical stimulations

Fig. 6. Application of EBFC culture to nanofibrous scaffolds. (a) SEM images of nanofibers made with either random or aligned structure. (b) Cell proliferation levels on the nanofiber scaffolds, counted at days 1 and 3, when cultured with or without EBFC-conditions. Statistically significant difference noticed for n ¼ 3, *; P < 0.05 vs. control, by ANOVA with Fisher post-hoc test. (c) Cell fluorescence images showing the cellular morphology in different culture conditions. Higher cell densities observed on EBFC-conditioned nanofiber scaffolds; furthermore, on the aligned nanofiber matrix, cells aligned and elongated with the underlying nanofibers, an observation even more evident with electrical stimulation, where more cells appeared to establish end-to-end contact.

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Fig. 7. Myogenic differentiation determined on the aligned nanofibrous scaffolds with or without EBFC-derived electrical stimulation. (a) RT-PCR gene expressions in different culture conditions on the nanofiber scaffolds. Band intensities also presented when normalized to GAPDH. (b) Immunofluorescence co-staining images of cells on the nanofiber scaffolds cultured under different conditions; myogenin in red, a-actinin in green and DAPI in blue. (c) Western blot analysis showing the protein expression levels of cells, and the band intensities quantified when normalized to b-actin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(Fig. 7a). In particular, the stimulation of genes appeared to be higher in the cathode and full-set conditions than in the anode condition (from the quantified band intensities). Next, myogenesis was examined at the protein level. The co-immunostaining of cells with anti-myogenin and anti-a-actinin antibody enabled the

visualization of protein localization and myotube formation under confocal imaging (Fig. 7b). Much stronger fluorescence signals were obtained from the cells cultured under electrical stimulations, and particularly from those cultured under full-set conditions. Furthermore, Western blot analysis revealed significantly higher

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band intensities of myogenin and a-actinin protein with electrical stimulations, particularly with full set enzyme loading (Fig. 7c). These observations in myogenic differentiation were quite similar to those in the 2D culture dish with EBFC stimulation. The only slight difference in the pseudo-3D scaffolding system compared to the 2D culture is that the stimulated outcomes manifested at a slightly later time point, i.e., a couple of days later in differentiation signals. This was thought to be because the underlying nanofiber substrates may require more time to form a tight anchorage of cells that allows for subsequent intracellular signals through the focal contact molecules, such as cytoskeletal rearrangement and differentiation signaling processes. Therefore, tuning the nanofiber composition to be more amenable to cellular anchorage including the tethering of adhesive molecules is considered a proper approach to be explored in further study. This will enable faster transmission of electrical signals to the intracellular compartments by enhancing the initial adhesion process. Even so, the EBFC-driven electrical stimulation of a series of events in C2C12 cells was well evidenced in the pseudo-3D systems. Therefore, the EBFC design is thought to be effectively translated to the implantable scaffolding system, which might enable more potential applications for the repair and regeneration of dysfunctional and diseased muscle tissues. While the current study demonstrates the in vitro functions of the EBFC-design and its combination with nanofibrous scaffolds, in vivo studies using proper disease models and damaged muscle tissues will potentiate the usefulness of the current system and warrants exploration in the near future. 4. Conclusions In this study, we demonstrate the development of a GOX and BOD-based enzyme biofuel cell (EBFC) for the purpose of muscle tissue repair and regeneration. The EBFC-generated electrical stimuli improved the proliferation, polarization and migration of muscle precursor cells in 2D culture. Significant stimulation of myogenic differentiation of cells was documented at the gene and protein levels. The EBFC-design could be easily integrated with pseudo-3D nanofibrous scaffolds where the underlying aligned nanotopographical cues can be combined with electrical signals for synergistic effects. Cellular proliferation and myogenesis were significantly stimulated by the novel EBFC-scaffold design. These results suggest the potential of integrating the EBFC-based electrical stimulation system with appropriate biomaterials and scaffolds for repair of damaged and dysfunctional muscle tissues. Furthermore, this system may prove valuable to other tissue engineering applications where bioelectrical signals play an important role. Acknowledgments This work was supported by the Priority Research Centers Program (grant#: 2009-0093829) through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology, South Korea. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.02.062. References [1] Liao IC, Liu JB, Bursac N, Leong KW. Effect of electromechanical stimulation on the maturation of myotubes on aligned electrospun fibers. Cell Mol Bioeng 2008;1:133e45.

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