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Functionalized Carbon Nanotube and Graphene Oxide Embedded Electrically Conductive Hydrogel Synergistically Stimulates Nerve Cell Differentiation Xifeng Liu,†,‡ A. Lee Miller II,‡ Sungjo Park,§ Brian E. Waletzki,‡ Zifei Zhou,† Andre Terzic,§ and Lichun Lu*,†,‡ †

Department of Physiology and Biomedical Engineering, ‡Department of Orthopedic Surgery, and §Department of Cardiovascular Diseases and Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota 55905, United States S Supporting Information *

ABSTRACT: Nerve regeneration after injury is a critical medical issue. In previous work, we have developed an oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel incorporated with positive charges as a promising nerve conduit. In this study, we introduced cross-linkable bonds to graphene oxide and carbon nanotube to obtain the functionalized graphene oxide acrylate (GOa) and carbon nanotube poly(ethylene glycol) acrylate (CNTpega). An electrically conductive hydrogel was then fabricated by covalently embedding GOa and CNTpega within OPF hydrogel through chemical cross-linking followed by in situ reduction of GOa in L-ascorbic acid solution. Positive charges were incorporated by 2-(methacryloyloxy)ethyltrimethylammonium chloride (MTAC) to obtain rGOaCNTpega-OPF-MTAC composite hydrogel with both surface charge and electrical conductivity. The distribution of CNTpega and GOa in the hydrogels was substantiated by transmission electron microscopy (TEM), and strengthened electrical conductivities were determined. Excellent biocompatibility was demonstrated for the carbon embedded composite hydrogels. Biological evaluation showed enhanced proliferation and spreading of PC12 cells on the conductive hydrogels. After induced differentiation using nerve growth factor (NGF), cells on the conductive hydrogels were effectively stimulated to have robust neurite development as observed by confocal microscope. A synergistic effect of electrical conductivity and positive charges on nerve cells was also observed in this study. Using a glass mold method, the composite hydrogel was successfully fabricated into conductive nerve conduits with surficial positive charges. These results suggest that rGOa-CNTpega-OPF-MTAC composite hydrogel holds great potential as conduits for neural tissue engineering. KEYWORDS: tissue engineering, nerve regeneration, conductive hydrogel, biodegradable polymer, graphene oxide, carbon nanotube, positive charge

1. INTRODUCTION There are approximately 100 000 patients that undergo neurosurgeries every year in the United States and Europe.1 However, nerve regeneration remains one of the most difficult issues in medical science as the number of nerve injuries caused by trauma and degenerative diseases continues to increase along with its associated healthcare burden.2,3 Nerve autograft, which aims to restore normal physiological structure through surgery, is the gold standard for clinical treatment of large nerve defects in peripheral nerve injuries. However, the limited graft availability and donor site morbidity drive the exploration for safer, readily available nerve conduits to repair nerve gaps. Advancements in the field of tissue engineering and biomaterial science have led investigators to develop synthetic nerve conduits as an alternative to nerve autograft. The ideal synthetic nerve conduits should have good biocompatibility not inducing adverse body reaction, excellent chemical structure that adapts to the physiologic environment, and suitable elasticity and © 2017 American Chemical Society

strength to allow regular motion of the muscles around the conduit without collapse of the tube. Biodegradable polymers are favorable materials for tissue regeneration benefiting from its self-disappearance through cleavage of covalent bond, thus leaving more space for tissue to grow in.4−8 Multiple natural polymers such as collagen, chitosan, and fibrin have been evaluated as nerve conduits for nerve regeneration.9−14 In recent years, synthetic polymers have immerged as promising candidates for nerve conduits, including polycaprolactone (PCL), poly(L -lactide acid) (PLLA), poly(caprolactone fumarate) (PCLF), poly(lactic-coglycolic acid) (PLGA), and oligo(poly(ethylene glycol) fumarate) (OPF).15−23 OPF is a biocompatible polymer synthesized from hydrophilic poly(ethylene glycol) (PEG) Received: February 13, 2017 Accepted: April 13, 2017 Published: April 13, 2017 14677

DOI: 10.1021/acsami.7b02072 ACS Appl. Mater. Interfaces 2017, 9, 14677−14690

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Figure 1. (a) Signal conduction in neuronal cells through polarization and depolarization of ionic channels. (b) Fluorescent images of PC12 cells on the neutral OPF hydrogel. (c) Schematic demonstration that PC12 cells proliferate slowly with minimal differentiation on neutral OPF hydrogel. (d) Incorporation of both conductive components and surface charges synergistically stimulates PC12 cell growth and the differentiation on rGOaCNTpega-OPF-MTAC composite hydrogel. (e) Fluorescent images of PC12 cells on the electrically conductive rGOaCNTpega-OPF-MTAC composite hydrogel. (f) Schematic demonstration that PC12 cells proliferate and differentiate on OPF hydrogel functionalized with electrical conductivity and positive charges.

Schwann cells, essential for neural tissue regeneration.26 These hydrogels were also reported to support excellent axonal growth for spinal cord repair in animal studies.32−36 In certain electroactive tissues, e.g., cardiovascular, neuronal, and muscle system, cells largely rely on the electrical conductivity (ion accumulation and flow) for coordinating cellular functions as well as signal transduction (Figure 1a).37−39 A series of recent studies have reported that introduction of electrical conductivity to underlying substrates could effectively improve neuronal cell behavior in terms of neurite and axon extension in in vitro investigations and nerve regeneration in animal work. 40−42 For example, after incorporation of conductive polypyrrole (PPy), the PLLA/ PPy, PLGA/PPy, chitosan/PPy, or cellulose/PPy conductive composite materials were reported to better stimulate neurite outgrowth and axon regeneration than the original nonconductive polymer. Conductive carbon materials, in the form of planar graphene sheets or linear nanotubes, were widely reported to stimulate nerve cell response.43,44 Our recent work has shown that the incorporation of chemically modified graphene oxide (GO) and carbon nanotubes (CNTs) could robustly enhance the growth of nerve cells and neurite extension from nerve cell bodies.45 In this study, we hypothesized that introducing positive surface charges together with embedding conductive carbon components within the OPF hydrogel could synergistically

components. In the presence of UV light or certain chemical reagents, the double bonds in OPF could open and cross-link with each other to form cross-linked hydrogel.24,25 The crosslinked OPF hydrogel is biodegradable by in situ hydrolysis.26,27 In our previous work, hollow tubes fabricated from OPF hydrogel were shown to effectively stimulate nerve regeneration in a rat sciatic nerve model.23 Electrical activities are common phenomena in certain biological tissues for preserving cell level homeostasis and intracellular development, repair, and tissue regeneration.28,29 Introduction of electric charges has been reported to stimulate proliferation and differentiation of nerve cells. For example, a greater extent of neurite outgrowth was observed on positively charged films than negatively charged and uncharged films, and neurite outgrowth correlated with the polarity and magnitude of the incorporated charges.30 The embryonic chick dorsal root ganglion (DRG) was also found to gain enhanced neurite extension on the polycationic chitosan with positive surface charges.31 In our previous study, OPF was cross-linked with [2(methacryloyloxy)ethyl]trimethylammonium chloride (MTAC) to form a positively charged OPF-MTAC hydrogel and showed enhanced effects on neuronal cell adhesion, proliferation, and differentiation during in vitro studies.26 The positively charged OPF hydrogels also largely enhanced the outgrowth of neurite from the dissociated DRG explant, which contains a combination of neurons, neuronal support cells, and 14678


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CNTpega-OPF-MTAC hydrogel conductive, these two types of hydrogels were reduced by soaking in excess L-ascorbic acid water solution (10 mg mL−1) for 4 days at 37 °C with gentle shaking. The reduced rGOa-CNTpega-OPF and rGOa-CNTpega-OPF-MTAC hydrogels were further washed in excess DI H2O for 3 days with water changed three times a day. 2.4. Hydrogel Characterization. Atomic Force Microscopy (AFM). Atomic force microscopy was utilized to investigate the morphology and layer height of the graphene oxides. The fabricated graphene oxide sample (∼1 μg/mL) was placed on the surface of freshly cleaved mica discs (Ted Pella, Redding, CA), incubated for several minutes, and then dried with nitrogen gas. Nanoscale AFM images (512 × 512 pixels) were collected in tapping mode using a Nanoscope IV PicoFroce Multimode AFM (Bruker) at room temperature.49,50 Swelling Ratio. OPF, OPF-MTAC, rGOaCNTpega-OPF, and rGOaCNTpega-OPF-MTAC hydrogels were dried under vacuum after chemical cross-linking. The dried materials were weighed (Wd) and swelled in DI H2O for 24 h and weighed (Ws) after removed of excess surface water. The swelling ratio was obtained using the equation swelling ratio = (Ws − Wd)/Wd. Differential Scanning Calorimetry (DSC). To evaluate thermal properties, dried composite hydrogels were analyzed by differential scanning calorimetry (DSC, TA Instruments) with specimens heated from to 0 to 200 °C under a constant heating rate of 10 °C min−1. Thermogravimetric Analysis (TGA). Thermal degradability of composite hydrogels was determined by thermogravimetric analysis (TGA, TA Instruments) with specimens heated to 700 °C under constant heating rate of 20 °C min−1. Scanning Electron Microscopy (SEM). Morphological structures of the four types of hydrogels dried by lyophilization were viewed on a scanning electron microscopy (SEM; S-4700, Hitachi Instruments, Tokyo, Japan). Transmission Electron Microscopy (TEM). The distribution of CNTpega tubes and reduced GOa sheets in these composite hydrogels was viewed by transmission electron microscope (TEM, 1200-EX II, JEOL Inc., Japan) at 80 kV voltage. The hydrogel samples were dried by lyophilization and buried into resin followed by a one-step cut into 0.6 μm thick films using a glass knife. A further cut was then made with a diamond knife into 0.1 μm layers before TEM examination. 2.5. Mechanical Properties and Conductivity. Hydrogels were punched into cylindrical specimens (∼12 mm in diameter and ∼0.8 mm in thickness). Compressive strain−stress curves were determined on a dynamic mechanical analyzer (DMA) (RSA-G2, TA Instruments) at a compression rate of 0.02 mm s−1 until failure. Compressive strengths and moduli of these hydrogels were calculated from the linear portion of the tested stress−strain curves. Conductivity of the four types of hydrogels was determined according to a previous study.45 Briefly, the resistance (R) of rectangular hydrogel samples immersed in Millipore DI water was read by a multimeter (34461A digital multimeter, 61/2 Digit, Keysight Technologies, Santa Rosa, CA), and electrical resistivity (ρ) was A W×H calculated by as ρ = R L = R L , where A indicates the crosssectional area of specimen, and L, H, and W indicate hydrogel length, height, and width, respectively. Electrical conductivity (σ) was calculated as the inverse of electrical resistivity, σ = 1/ρ, with unit of siemens per meter (S/m). 2.6. Cytotoxicity. In order to elute the cross-linking agents and sol fractions, the cross-linked hydrogels were emerged in DI H2O for 2 days with three water changes, followed by sterilization in 70% alcohol solution for 24 h and subsequent washing with sterilized phosphate buffered saline (PBS) at least five times in 3 days. The PC12 cells were purchased from ATCC and used for the biological compatibility test. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% horse serum, 5% fetal bovine serum, and 0.5% streptomycin/penicillin and placed in the cell culture incubator with the parameters set as 95% relative humidity, 5% CO2, and 37 °C. After trypsinization, PC12 cells were resuspended in DMEM, counted, and seeded at a concentration of 30 000 cells cm−2

stimulate nerve cell responses. This concept is illustrated in Figures 1b−f. OPF, GO sheets functionalized with acrylate (GOa), CNTs functionalized with PEG acrylate (CNTpega), and MTAC were chemically cross-linked, followed by in situ reduction of GOa to obtain the final electrically conductive, positively charged rGOa-CNTpega-OPF-MTAC hybrid hydrogel. The synthesized hydrogel was characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM), and thermogravimetric analysis (TGA). The mechanical strength, thermal properties, and distribution of rGOa/CNTpega carbon contents in the hydrogel were characterized by dynamic mechanical analysis (DMA), differential scanning calorimetry (DSC), and transmission electron microscopy (TEM), respectively. Electrical resistance, cytotoxicity, and effectiveness of the hydrogel in stimulating nerve cell proliferation, spreading, and neuronal differentiation were evaluated.

2. MATERIALS AND METHODS 2.1. Synthesis of CNTpega. To functionalize carbon nanotubes, CNT-COOH was reacted with excess HO-PEG-OH polymer using dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP) at 120 °C for 24 h.45 The resulting CNTpeg was further reacted with acryloyl chloride in the presence of excess triethylamine (Et3N) for 2 days under nitrogen at room temperature to introduce the cross-linkable acrylate functional groups. The final functionalized CNTpega was collected by precipitation into excess acetone for two times and ethanol two times, then freeze-dried, and stored at −20 °C for usage. 2.2. Synthesis of GOa. Graphene oxide (GO) was synthesized by the improved Hummers’ method using graphite flakes.46 Purified GO sheets were then reacted with acrylolyl chloride in N,N-dimethylformamide (DMF) in the presence of excess Et3N as a proton scavenger to introduce cross-linkable double bonds to the surface, as reported previously.45 Obtained GOa was precipitated in acetone, dialyzed within distilled deionized (DI) water for 3 days in a cellulose dialysis bag (MWCO 2000), and dried under vacuum. 2.3. Fabrication of Composite Hydrogels. The OPF polymer was obtained using previously reported synthetic routes.47,48 Before cross-linking, OPF polymer (1 g) and N,N′-methylenebis(acrylamide) (BIS, 36 mg) were fully dissolved in DI H2O (2 mL) and mixed on a vortex. Ammonium persulfate (APS) solution was prepared by dissolving 0.5 g of APS in 1 mL of DI H2O, and N,N,N′,N′tetramethylethylenediamine (TEMED) solution was prepared by adding 0.5 mL of TEMED in 1 mL of H2O. For hydrogel fabrication, 0.1 mL of prepared APS solution and 0.1 mL of TEMED solution were added to the OPF/BIS mixture and transferred immediately to silicone rubber molds with 0.8 mm thickness. The mold was then covered by two glass slides, and the hydrogel was allowed to fully cross-link for 1 h in a 60 °C oven. To fabricate the positively charged OPF-MTAC hydrogel, 1 g of OPF, 36 mg of BIS, 30 μL of MTAC solution, and 2 mL of DI H2O were mixed and cross-linked by APS and TEMED following the same steps as described above. For fabricating conductive hydrogels, functionalized GOa sheets (0.05 g) were distributed in 5 mL of DI H2O using a probe model sonicator (Qsonica Q500). After that, the functionalized CNTpega (0.005 g) were then added and further sonicated to distribute the CNTpega in the solution. 1 mL of the prepared GOa/CNTpega mixture was vortexed with OPF (0.5 g) and BIS (18 mg), further sonicated using the probe model sonicator until a homogeneous distribution of carbon contents, and then cross-linked by the addition of APS/TEMED using the same procedure described above. To obtain the positively charged GOa-CNTpega-OPF-MTAC hydrogel, 1 g of OPF, 36 mg of BIS, and 30 μL of MTAC solution were mixed with 2 mL of GOa-CNTpega solution, sonicated, and chemically cross-linked as described above. To remove the sol fraction and impurities, all four types of crosslinked hydrogels were washed in excess DI H2O for 2 days with at least four water changes. To make the GOa-CNTpega-OPF and GOa14679


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Figure 2. Synthesis and characterization of CNTpega and GOa. (a) Synthesis procedures for obtaining CNTpega carbon material. SEM observation of (b) CNT-COOH and (c) CNTpega after reaction. TEM images of (d) CNT-COOH and (e) CNTpega after reaction. (f) Synthesis route of GOa and (g) AFM images showing morphology and layer height of GOa after reaction. in 12-well tissue culture polystyrene (TCPS) plates. After 24 h of cell attachment, transwell chambers (mesh size, 3 μm) containing different composite hydrogels were placed into various wells. After 4 days of coculture, WST-1 assay (Roche Applied Science, Mannheim, Germany) was performed to quantify the cell number. The absorbance value was read at 450 nm by an ultraviolet−visible absorbance microplate reader (SpectraMax Plus 384, Molecular Devices, Sunnyvale, CA). The cell viability in the wells without hydrogels was set as 100% and used to determine the corresponding cell viability in the hydrogel-treated wells. 2.7. Proliferation and Spreading of PC12 Cells on Hydrogels. After cross-linking, hydrogels were washed and sterilized as described above. The hydrogels were shaped into round disks by punching with a cork borer (diameter 13.2 mm). Hydrogel disks were firmly set on the bottom of 12 well tissue culture plates using autoclave sterilized vacuum grease (Dow Corning, Midland, MI). The hydrogels were presaturated with DMEM medium for 2 h before cell seeding at a concentration of 30 000 cells cm−2. After 1, 4, and 7 days of incubation in DMEM supplemented with 10% horse serum, 5% fetal bovine serum, and 0.5% streptomycin/penicillin, cell numbers were quantified by the MTS assay. To observe morphological features of cells on these hydrogels, attached PC12 cells were fixed with a 4% paraformaldehyde (PFA) solution after 4 days postseeding. After permeabilization by 0.2% Triton X-100 solution, cells were stained for 1 h at 37 °C with antivinculin−FITC antibody (Sigma-Aldrich Co., Milwaukee, WI) to

stain the cellular focal adhesions. After that cells were further stained with rhodamine−phalloidin (RP, red) to visualize F-actin arrangement and finally 10 min with 4′,6-diamidino-2-phenylindole (DAPI, blue) to visualize cell nuclei. The fluorescent images of stained cells were taken on an inverted laser scanning confocal microscope (Carl Zeiss, Germany). 2.8. Neuronal Differentiation of PC12 Cells on Hydrogels. To investigate neurite development, PC12 cells were seeded on the four types of hydrogels at a density of 30 000 cells cm−2 and cultured in DMEM supplemented with 10% horse serum, 5% fetal bovine serum, and 0.5% streptomycin/penicillin for 12 h to allow attachment. Then the medium was removed, and hydrogels were washed three times using sterilized PBS to remove unattached cells. Cells on hydrogels were then induced using DMEM medium supplemented with 1% FBS and 50 ng mL−1 of nerve growth factor (NGF) for 4 days in the incubator. Differentiated PC12 cells were fixed with a 4% paraformaldehyde (PFA) solution and permeabilized by 0.2% Triton X-100 solution. Immunostaining was conducted in sequence with antivinculin−FITC antibody to stain the cellular focal adhesions, rhodamine−phalloidin (RP, red) to visualize F-actin arrangement, and DAPI to visualize cell nuclei using the same protocol as described above. The fluorescent images of stained cells were taken on an inverted laser scanning confocal microscope (Carl Zeiss, Germany). 2.9. Nerve Conduit Fabrication. A glass tube mold with a stainless steel rod inside was manufactured and assembled and 14680


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Figure 3. (a) Fabrication of conductive composite GOaCNTpega-OPF-MTAC hydrogel and in situ reduction of GO sheets in L-ascorbic acid solution. (b) Photographs of fabricated (i) OPF, (ii) OPF-MTAC, (iii) rGOaCNTpega-OPF, and (iv) rGOaCNTpega-OPF-MTAC hydrogels. (c) Swelling ratios of the four types of hydrogels fabricated. centered by a polytetrafluoroethylene (PTFE) spacer ring at both ends. The aqueous GOa/CNTpega/OPF/MTAC mixture solution with APS and TEMED were injected to fill the space between the rod and the outer glass mold. The mold together with polymer resin was placed in 60 °C oven for 1 h to allow full cross-linking of the components. Afterward, the GOaCNTpega-OPF-MTAC tube was removed from the mold and placed in 10 mg mL−1 L-ascorbic acid solution at 37 °C with gentle shaking for 4 days to allow reduction of GOa sheets to generate conductive rGOaCNTpega-OPF-MTAC hydrogel conduit. 2.10. Statistical Analysis. Data analysis was performed by oneway analysis of variance (ANOVA) and further Tukey post-test if necessary. Differences are considered significant when p-values are smaller than 0.05.

both CNT-COOH and CNTpega ranges from 10 to 30 nm, with CNTpega slightly larger in diameter (Figures 2b,c). TEM imaging at higher magnification demonstrated a dark layer surrounding CNTs after reaction, indicating the pega polymer was successfully incorporated to the CNT surface (Figures 2d,e). The synthesis route of cross-linkable GOa sheets from GO precursors was shown in Figure 2f. After acrylation, an obvious acrylate peak due to double bonds (CC, wavenumber ∼1649 cm−1) on the cross-linkable GOa sheets was detected, indicating successful incorporation of cross-linkable double bonds to the GO sheets.45 AFM images showed a detectable layer ∼1 nm in thickness for the GOa sheets, indicating successful exfoliation of GOa into single layered sheets (Figure 2g). 3.2. Morphological and Mechanical Properties of Composite Hydrogel. Four types of hydrogels were fabricated with the addition of surface charge or GOa/ CNTpega carbon composites (Figure 3a). For neutral OPF hydrogels, neither surface charge nor conductivity was incorporated. As shown in Figure 3b, the neutral OPF hydrogel disks are transparent and mainly used as a control for the other three types of functionalized hydrogels. The second type of hydrogel was fabricated by cross-linking small MTAC molecules into the OPF network. The resulting OPF-MTAC is transparent with positive charges provided by the pendant

3. RESULTS AND DISCUSSION 3.1. Covalently Functionalized CNTpega and GOa. To functionalize CNTs, PEG chains were grafted through an esterification reaction with carboxyl groups in CNT (Figure 2a). Cross-linkable CNTpega was further synthesized by reacting with acryloyl chloride. After reaction, typical PEG chain peak (wavenumbers 2828−2890 cm−1) and acrylate groups peak (wavenumbers around 1649 cm−1) were detected by ATR-FTIR analysis.45 As shown by SEM, the CNT-COOH before reaction and CNTpega obtained both exhibited tubular morphological features (Figures 2b,c). The size distribution of 14681


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Figure 4. (a) Compressive stress−strain curve and (b) compressive modulus of the four types of hydrogels were characterized. SEM images of (c) OPF, (d) OPF-MTAC, (e) rGOaCNTpega-OPF, and (f) rGOaCNTpega-OPF-MTAC hydrogels after drying by lyophilization.

Figure 5. TEM images of thin layers of (a) OPF, (b) OPF-MTAC, (c) rGOaCNTpega-OPF, and (d) rGOaCNTpega-OPF-MTAC hydrogels. (e) Schematic representation of carbon contents observed by TEM for rGOaCNTpega-OPF and rGOaCNTpega-OPF-MTAC hydrogels. (f) DSC and (g) TGA curves of the four types of hydrogels in the dried state. (h) Conductivity of pure Millipore water and the four types of hydrogels in the hydrated state.

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Figure 6. Live (green) and dead (red) viability analysis of PC12 cells on (a) OPF, (b) OPF-MTAC, (c) rGOaCNTpega-OPF, and (d) rGOaCNTpega-OPF-MTAC hydrogels. (e) Number of live and dead cells calculated from cellular fluorescence images on the four types of hydrogels. (f) Cell viability as determined using WST-1 assay after coculture of PC12 cells with the four types of hydrogels for 4 days.

MTAC chains (Figure 3b). To investigate the effect of conductivity, a third type of hydrogel was fabricated by incorporating cross-linkable GOa sheets together with functionalized CNTpega tubes within the polymeric network. GOa sheets were then reduced by L-ascorbic acid solution. As shown in Figure 3b, the fabricated rGOaCNTpega-OPF hydrogel are opaque with dark color resulting from the dark carbon compositions in the hydrogel. The fourth type of hydrogel combines the positive charge together with conductivity to generate a novel rGOaCNTpega-OPF-MTAC hydrogel, which adopts a similar dark color to the rGOaCNTpega-OPF hydrogel. In the rGOaCNTpega-OPF-MTAC hydrogel, positive charge was provided by the pendant MTAC molecules whereas the conductivity was originated from the reduced GOa sheets and the interconnecting CNTpega tubes. After cross-linking, the swelling of all four types of hydrogels in DI water was characterized. It is noticeable from Figure 3c that the incorporation of either MTAC chain or GOa sheets restricted the swelling of OPF chains. This can be evidenced by the highest swelling ratio for pure OPF hydrogel of 12.4 ± 1.2 and reduced swelling ratio for OPF-MTAC hydrogel of 10.6 ± 1.5 and rGOaCNTpega-OPF hydrogel of 11.8 ± 1.5 times the original network. For the rGOaCNTpega-OPF-MTAC composite hydrogel with both MTAC chains and GOa/CNTpega carbon contents, the lowest swelling ratio was detected to be 8.7 ± 0.5. The mechanical properties of the four types of hydrogels were characterized by compressive DMA tests. The compressive strain−stress curves were combined and presented in Figure 4a. An obvious trend can be found that the cross-linking of MTAC small molecules or carbon contents into the OPF hydrogel largely increased the mechanical strength of the hydrogel. The compressive moduli calculated from the linear

slope of the compressive strain−stress curve showed increasing values of 100.6 ± 53.3, 159.9 ± 82.0, 538.0 ± 287.5, and 850.5 ± 670.5 kPa for OPF, OPF-MTAC, rGOaCNTpega-OPF, and rGOaCNTpega-OPF-MTAC hydrogels, respectively (Figure 4b). The enhancement of mechanical strength and restriction of swelling for composite hydrogel are mainly due to the tightening of the OPF network caused by the small cross-linker MTAC chain and cross-linkable GOa/CNTpega carbon contents. SEM images of the cutting edges of all four types of hydrogels are presented in Figures 4c−f. The neutral OPF hydrogel and the positively charged OPF-MTAC hydrogel showed similar structure with solidified internal layers. However, for the rGOaCNTpega-OPF and rGOaCNTpegaOPF-MTAC hydrogel incorporated with carbon contents, a unique porous structure was demonstrated with more empty spaces inside the hydrogel substrates. These morphological features were largely caused by the strong strength of GOa/ CNTpega carbon contents, which provided mechanical support and resistance to structural deformation of the hydrogel. 3.3. Distribution of GOa and CNTpega in Hydrogel and Conductivity. The distribution of GOa/CNTpega carbon compositions inside these conductive hydrogels was examined by TEM. As can be seen from Figures 5a,b, OPF and OPF-MTAC hydrogels showed high transparency with the absence of GOa/CNTpega under TEM. In comparison, rGOaCNTpega-OPF and rGOaCNTpega-OPF-MTAC hydrogels were viewed with dark color, which was resulted from the GOa/CNTpega carbon compositions (Figure S1). Enlarged view demonstrated adispersed distribution of GOa sheets/ CNTpega tubes within the hydrogels without forming significant aggregates (Figures 5c,d). A schematic demonstration of GOa sheets and CNTpega tubes in rGOaCNTpega14683


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Figure 7. Cellular F-actin (red), vinculin (green), nuclei (blue), and merged fluorescent images of PC12 cells on (a) OPF, (b) OPF-MTAC, (c) rGOaCNTpega-OPF, and (d) rGOaCNTpega-OPF-MTAC hydrogels at 4 days postseeding. (e) MTS absorbance of PC12 cell at 1, 4, and 7 days postseeding on the four types of hydrogels. The distribution and average value of (f) cellular spreading area and (g) circularity calculated using 50 single cells at 4 days postseeding on the four types of hydrogels. (h) Percent of neurite bearing PC12 cells at 4 days postseeding on these hydrogels.

curves presented in Figure 5f. This may be because the rGOa sheets and CNTpega tubes partially inhibited the crystallization of the polymer chains. Thermal degradation of the four types of hydrogel was analyzed by TGA testing from room temperature to 700 °C. As demonstrated in Figure 5g, a weight loss

OPF and rGOaCNTpega-OPF-MTAC hydrogel is shown in Figure 5e. Thermal analysis determined by DSC tests showed a slight decrease in melting temperatures after incorporation of rGOa and CNTpega carbon contents, as seen from the heat flow 14684


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ACS Applied Materials & Interfaces occurred when the temperature rose to 400 °C, and the final weights remaining for the four types of hydrogel were all below 10% of the original mass. Because of the presence of rGOa and CNTpega carbon contents, rGOaCNTpega-OPF and rGOaCNTpega-OPF-MTAC hydrogels had slightly more mass left after degradation of polymer compositions when temperature increased to 700 °C. After removal of impurities in the Millipore deionized water for 3 days, the hydrogel conductivity was tested separately, using the pure deionized water as a control. The conductivity of the deionized water was around (4.16 ± 0.41) × 10−5 S/m, which is close to the previous assessment of 7.5 × 10−5 S/m for the deionized water upon fully equilibration to the atmosphere.51 A small quantity of carbonic acid caused by dissolution of CO2 from air into water might explain the slight conductivity of the deionized water. As shown in Figure 5h, the neutral OPF hydrogel and positively charged OPF-MTAC hydrogel had conductivity of (3.16 ± 1.39) × 10−4 and (6.24 ± 2.70) × 10−4 S/m, respectively. The slight increase in conductivity of OPFMTAC hydrogel is mainly because the MTAC molecule provided possible free ions to the composite hydrogel. For rGOaCNTpega-OPF hydrogel, the conductivity was tested to have a significantly increased value of (2.96 ± 1.86) × 10−3 S/ m. This enhancement was mainly contributed by the conductive rGOa and CNTpega carbon components. For the rGOaCNTpega-OPF-MTAC hydrogel with both charges and carbon compositions, the conductive had the highest value of (5.75 ± 3.23) × 10−3 S/m due to the synergistic contribution from MTAC molecules and rGOa and CNTpega carbon components. The hydrogels embedded with only rGOa or CNTpega showed reduced conductivity compared to the rGOaCNTpega-OPF-MTAC hydrogel with both rGOa and CNTpega carbon contents (Figure S2a). This may be due to better interconnections in the rGOaCNTpega-OPF-MTAC hydrogel compared with the rGOa-OPF-MTAC or CNTpegaOPF-MTAC hydrogel with only one component (Figure S2c). However, it is very important to note that the conductivity varies by the distribution of carbon contents inside the hydrogel. It is believed that the polymer chains could cause the gathering of CNTpega and rGOa, as evidenced by high conductivity of GOa/CNTpega mixture in water which was reduced after addition of the polymers to the mixture before cross-linking. Therefore, a vigorous sonication process is required to fully distribute the carbon contents in the mixture followed by immediate cross-linking. 3.4. Cytotoxicity of Composite rGOaCNT-OPF-MTAC Hydrogels. The cytotoxicity of these hydrogels to PC12 cells was evaluated by both live/dead cell assays and MTS tests. As noted in Figure 6a, the live cells on OPF hydrogel were stained with green fluorescence whereas the dead cells were stained red. Compared with neutral OPF hydrogel, the positively charged OPF-MTAC and conductive rGOaCNTpega-OPF hydrogel showed more green fluorescent cells, as seen in Figures 6b,c. For synergistically functionalized rGOaCNTpegaOPF-MTAC hydrogels with charge and conductivity, the most densely distribution profile of PC12 cells was observed (Figure 6d). Quantitative analysis of live/dead cell numbers on these four types of hydrogel showed highest number of PC12 cells on the rGOaCNTpega-OPF-MTAC hydrogel with the lowest number of cells on neutral OPF hydrogel (Figure 6e). To confirm the results, an alternative WST-1 test was conducted. As can be seen from Figure 6f, all the four types of hydrogel showed virtually no cytotoxicity to cells. The good biocompat-

ibility of the rGOaCNTpega-OPF-MTAC composite hydrogel makes it a promising material for tissue engineering applications. 3.5. Proliferation of PC12 Cells on Hydrogels. The proliferation, spreading, and neurite development of PC12 cells on these hydrogels were investigated. The cellular growth on the four types of hydrogels as well as TCPS positive control was detected by MTS. At 1, 4, and 7 days postseeding of PC12 cells to the hydrogels, the optical absorbance of MTS assay was determined using a microplate reader. As seen from Figure 7e, the TCPS positive control showed the highest value whereas the lowest OD absorbance appeared for the neutral OPF hydrogel. Compared with the neutral OPF hydrogel, the conductive rGOaCNTpega-OPF and rGOaCNTpega-OPFMTAC hydrogel showed significantly higher adsorption at 4 days postseeding. At 7 days postseeding, the other three functionalized hydrogels all showed significantly higher OD values than the neutral OPF hydrogel with the highest value demonstrated for the charged and conductive rGOaCNTpegaOPF-MTAC hydrogel. The rGOaCNTpega-OPF-MTAC hydrogel with both rGOa and CNTpega carbon contents also had better cell proliferation than hydrogels embedded with only rGOa or CNTpega (Figure S2b). At 4 days postseeding, the cells were fixed, stained, and photographed. The typical fluorescence images of PC12 cells at 4 days postseeding are presented in Figures 7a−d. The cellular F-actin was stained with rhodamine−phalloidin (red) and cell nuclei with DAPI (blue), and a merge of the two components in different colors was also provided to make clearer comparison of cell morphologies on these hydrogels. An obvious trend can be seen from the images that higher density of cells was attached to the OPF-MTAC and rGOaCNTpegaOPF and rGOaCNTpega-OPF-MTAC hydrogel than the neutral OPF hydrogel (Figures 7a−d). Furthermore, cells spread to larger area on the functionalized hydrogels than the neutral OPF hydrogel. Clear neurite extension can be found for cells on the rGOaCNTpega-OPF-MTAC hydrogel synergistically functionalized with surface charges and inherent conductivities compared to the neutral OPF hydrogel. The cellular spreading, circularity, and percent of neurite bearing cells on these hydrogels were analyzed. The cell area distribution of 50 single cells on these four types of hydrogels were analyzed and averaged. As shown in Figure 7b, cells on neutral OPF hydrogels showed limited spreading with average cell area of 934 ± 398 μm2. With positive charge stimulation, cells on the OPF-MTAC hydrogel had elevated spreading with average cell area of 1135 ± 744 μm2. Conductivity properties also enhanced the spreading of cells, as evidenced by more spreading of cells on conductive rGOaCNTpega-OPF hydrogel with average spreading area of 1119 ± 639 μm2. For the fourth type of hydrogel that owns both charge and conductive properties, cells showed a higher spreading area with average value of 1522 ± 629 μm2, indicating that surface charge and inherent conductivity could synergistically enhance the cell functions. Circularity of cells on the hydrogels was also analyzed. Circularity, as defined by the equation of 4π × area/perimeter2, is used to describe how close the cellular shape approaches that of a round circle.52 For cells with shapes tend to be round, the circularity is closer to 1.0. On the contrary, for cells with more linear shapes, the circularity value is closer to 0.0. As noted in Figure 7c, cells on neutral OPF hydrogel have a circularity distribution closer to 1.0 with average values of 0.59 ± 0.16, 14685


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Figure 8. Single cellular F-actin (red), vinculin (green), and nuclei (blue) and merged fluorescent images of PC12 cells after induced differentiation on the (a) OPF, (b) OPF-MTAC, (c) rGOaCNTpega-OPF, and (d) rGOaCNTpega-OPF-MTAC hydrogels at 4 days postseeding. (e) The neurite length distribution and average value for 100 independent neurites developed in cells growing on these hydrogels. (f) The elongation of nuclei in cells on the four types of hydrogels. (g) Enlarged view of focal adhesion development in the PC12 cells differentiated on the four types of hydrogels. Schematic demonstration of cellular behavior on (h) neutral OPF hydrogel and (i) rGOaCNTpega-OPF-MTAC hydrogel with surface charges and improved conductivity.

indicating a more round-like shape for cells on the hydrogel. After introducing of charges or conductivity to the hydrogel, a reduced circularity with values of 0.51 ± 0.20 and 0.52 ± 0.18 were determined for OPF-MTAC and rGOaCNTpega-OPF hydrogel, respectively. The lowest circularity distributions were detected for cells on positively charged conductive rGOaCNTpega-OPF-MTAC hydrogel with an average value of 0.45 ± 0.17, implying a more linear shape compared with the neutral OPF hydrogel. The percent of neurite bearing cells on the four types of hydrogels was also calculated. As shown in Figure 7d, on neutral OPF hydrogels, approximately 17.1 ± 6.2% of cells had developed neurites. However, with the introduction of charge or conductivity properties, the functionalized hydrogels showed significantly higher percentage of neurite bearing cells than the neutral OPF hydrogels with values of 31.0 ± 5.2% and 32.4 ± 6.7% for OPF-MTAC and rGOaCNTpega-OPF hydrogel,

respectively. Owning synergistically functionalized surface charge and inherent conductivity, the rGOaCNTpega-OPFMTAC composite hydrogel showed the highest percent of neurite bearing cells with a value of 42.2 ± 7.3%. This trend demonstrated that the surface charge and inherent conductivity could robustly promote neuronal differentiation of PC12 cells on the OPF hydrogels. 3.6. Neuronal Differentiation of PC12 Cells on Hydrogel. After induced differentiation using 50 ng mL−1 NGF, each nonoverlapping single PC12 cell on these hydrogels is presented in Figures 8a−d. The neurite lengths developed from cell bodies were determined using the ImageJ software. As can be seen in Figure 8e, the distribution profile of lengths for 100 independent neurites on the four types of hydrogels showed apparent differences. The neurites in cells on neutral OPF hydrogel all gathered within 50 μm with an average value of 9.9 ± 8.7 μm. For OPF-MTAC and rGOaCNTpega-OPF 14686


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Figure 9. Nerve conduit fabrication and characterization. (a) Schematic demonstration of the tubular conduit fabrication process. (b) Photographs of the fabricated tubular conduit. (c) SEM images of freeze-dried conduits at different angles. (d) Schematic view of conduit mediated stimulation of nerve growth aiming at repair of damaged nerves.

hydrogels, there are a few neurites that grew to lengths over 50 μm with average value of 14.4 ± 13.0 and 13.9 ± 11.0 μm, respectively. A perceivable number of neurites with lengths over 50 μm were developed in cells on rGOaCNTpega-OPF-MTAC hydrogel with average value of 16.3 ± 15.6 μm (Figure 8e). The circularity of each cellular nuclear was calculated using ImageJ software, and then the elongation of nuclei was further obtained using the inverse of given nuclear circularity.52 The results showed more stretched elongated cellular nuclei for cells on the conductive rGOaCNTpega-OPF hydrogel (Figure 8f). Enlarged view of the neurite protrusion parts displayed a better extended morphology for cells on either positive charged OPF-MATC hydrogel or conductive rGOaCNTpega-OPF hydrogel compared with the neutral OPF hydrogel (Figure 8g). However, the conductive and positively charged rGOaCNTpega-OPF hydrogel showed the most robust development of neurites comparted to the other counterparts. In addition, focal adhesion developments were also demonstrated to be better on the three conductive or positively

charged composite hydrogels compared to the neutral OPF hydrogel (Figure 8g). The potential mechanism for the stimulated neurites on the rGOaCNTpega-OPF-MTAC hydrogel compared with the neutral OPF hydrogel is schematically demonstrated in Figures 8h,i. On the neutral OPF hydrogel, cells could not develop good adhesion sites and spreading on the surface due to poor cell/matrix interaction caused by the repelling effects of the PEG chains and lack of supportive components (Figure 8h). In contrast, on the rGOaCNTpega-OPF-MTAC hydrogel, the reduced GOa sheets and functionalized CNTpega carbon nanotubes provided stronger mechanical strength, thus a stiffer surface for cells to adhere and spread (Figure 8i). Further, neuronal cells were widely acknowledged to favor conductive surfaces because the conductive underlying substrates have the possibility to help the electrical stimulation within cells and among adjacent cells.53 The interconnected rGOa/CNTpega carbon components built a conductive network for the PC12 cells to communicate on rGOaCNTpega-OPF-MTAC hydro14687


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facilitate cellular function and conducting of electrical signals for the electroactive neuronal cells. On the basis of good biocompatibility and excellent in vitro cell proliferation and differentiation, the novel rGOaCNTpega-OPF-MTAC hydrogel developed in this study is expected to have promising potential for peripheral nerve repair applications. The conduits fabricated by rGOaCNTpega-OPF-MTAC hydrogel owns positive charged surfaces and inherent incorporated conductivity, which are unique advantages for function as nerve conduits. Controllable approach of neuronal differentiation is believed to provide more options for clinical applications in neuronal regeneration.64 Further animal studies are underway to investigate axon growth under in vivo conditions for the rGOaCNTpega-OPF-MTAC hydrogel system.

gel therefore enhanced the cellular behaviors including spreading, proliferation, and differentiation (Figure 8i). For electroactive cells that frequently require electric activities for contraction or signal conduction, e.g., cardiac, neuron, muscle, or even bone cells, they largely rely on the electrical conductivity for realizing certain cellular functions.37−39 The electrical stimulation could enhance osteogenic differentiation of human bone marrow mesenchymal stem cells, indicating the potential use of conductive substrate for bone regeneration. 54 Heart muscles were reported to have approximate conductivity of 0.1 S/m and supported by electrically conductive fibers.55,56 Recent research reported the introduction of small amount of CNTs formed an electrically conductive hydrogel strongly improved adhesion of cardiac cell and enhanced intercellular electrical coupling.57 Nerve tissues were reported to have conductivities in the range from 1.0 × 10−2 to 5.7 × 10−1 S/m level, depending on the test method, nerve types, and frequencies applied.58−60 The adhesion and proliferation of both cardiomyocytes and neuroblastoma cells were reported to be modulated through changing the underlying substrates’ conductivities using conductive carbon-nanofiber/PLGA composite or directly using hydroxyapatite−calcium titanate (HA-CaTiO 3 ) model.61,62 It is also reported that the electrical signal from graphene-based electrodes could stimulate mesenchymal stem cells into Schwann-like cells.63 Previous work in our lab demonstrated that the incorporation of conductive polypyrrole (PPy) or carbon contents into substrates strongly stimulated the adhesion and proliferation of neuronal PC12 cells.40,41,45 In this study, we combined the conductive carbon contents and positive charges to fabricate the rGOaCNTpega-OPF-MTAC hydrogel. The conductivity was tested to be around (5.75 ± 3.23) × 10−3 S/m, which is close to that reported for native nerve tissue. Enhanced effects were observed extensively for cellular attachment, proliferation, differentiation, and focal adhesion development, consistent with the previous work mentioned above. 3.7. Nerve Conduit Fabrication. Tubular rGOaCNTpegaOPF-MTAC hydrogel conduits were fabricated by a molding method with detailed steps demonstrated in Figure 9a. The cross-linkable GOa/CNTpega/OPF/MTAC formulation was combined with APS as chemical initiator and TEMED as accelerator and injected into a glass mold assembled with a stainless steel rod in the center. The cross-linking occurred inside the tube mold after an hour of incubation at 60 °C. The obtained tube was then reduced in 10 mg mL−1 L-ascorbic acid solution at 37 °C for 4 days to generate conductive rGOaCNTpega-OPF-MTAC hydrogel conduit. Photographs of the fabricated rGOaCNTpega-OPF-MTAC conduit showed a homogeneous diameter and wall thickness, as can be seen in Figure 9b. The conduit had sufficient mechanical strength allowing easy handling using tweezers. The structural morphology of the conduit after freeze-drying under vacuum was examined by SEM. The images showed that the conduit had a homogeneous wall thickness and similar morphological features at cross sections (Figure 9c). These results indicated that the molding method was an effective route for fabrication of rGOaCNTpega-OPF-MTAC hydrogel into nerve conduits. For nerve regeneration, the conduits applied should provide guidance for the regenerating axons to grow across the defect reaching the distal end. In addition, for the electrically conductive tissue, inserted implants are expected to have sufficient electrical conductivity in order to help

4. CONCLUSIONS An electrically conductive hydrogel was fabricated which is composed of reduced graphene oxide sheets and functionalized carbon nanotubes for nerve regeneration applications. To further synergistically enhance nerve cell functions, small molecular MTAC chains were also incorporated providing positive charges to the composite hydrogel. The final obtained rGOa-CNTpega-OPF-MTAC composite hydrogel with surface charges and electrical conductivity showed good biocompatibility and excellent enhancement for PC12 cell proliferation and spreading. Neurite development of PC12 cells was also observed to be largely stimulated on the rGOa-CNTpega-OPFMTAC composite hydrogel compared with the neutral OPF hydrogel. Using a molding method, these hydrogels were successfully fabricated into conductive nerve conduits with surficial positive charges. These results demonstrated promising potential for the rGOa-CNTpega-OPF-MTAC composite hydrogel to serve as conduits for neural tissue engineering.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b02072. TEM images of thin cut layers of cross-linked OPF, OPF-MTAC, rGOaCNTpega-OPF, and rGOaCNTpegaOPF-MTAC hydrogels (Figure S-1); electrical conductivities and normalized cell proliferation on OPFMTAC, CNTpega-OPF-MTAC, rGOa-OPF-MTAC, and rGOaCNTpega-OPF-MTAC hdyrogels at 7 days postseeding, with OPF-MTAC set as 100% (Figure S-2); schematic demonstration of differences in carbon content distribution in rGOa-OPF-MTAC, CNTpega-OPFMTAC, and rGOaCNTpega-OPF-MTAC hdyrogels (Figure S-2) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(L.L.) E-mail [email protected]; Ph 507-284-2267; Fax 507-284-5075. ORCID

Xifeng Liu: 0000-0002-2684-6309 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Mayo Foundation. 14688


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(18) Schnell, E.; Klinkhammer, K.; Balzer, S.; Brook, G.; Klee, D.; Dalton, P.; Mey, J. Guidance of glial cell. migration and axonal growth on electrospun nanofibers of poly-epsilon-caprolactone and a collagen/poly-epsilon-caprolactone blend. Biomaterials 2007, 28 (19), 3012−3025. (19) de Ruiter, G. C.; Spinner, R. J.; Malessy, M. J. A.; Moore, M. J.; Sorenson, E. J.; Currier, B. L.; Yaszemski, M. J.; Windebank, A. J. Accuracy of motor axon regeneration across autograft, single-lumen, and multichannel poly(lactic-co-glycolic acid) nerve tubes. Neurosurgery 2008, 63 (1), 144−153. (20) Yao, L.; Wang, S. G.; Cui, W. J.; Sherlock, R.; O’Connell, C.; Damodaran, G.; Gorman, A.; Windebank, A.; Pandit, A. Effect of functionalized micropatterned PLGA on guided neurite growth. Acta Biomater. 2009, 5 (2), 580−588. (21) Koh, H. S.; Yong, T.; Teo, W. E.; Chan, C. K.; Puhaindran, M. E.; Tan, T. C.; Lim, A.; Lim, B. H.; Ramakrishna, S. In vivo study of novel nanofibrous intra-luminal guidance channels to promote nerve regeneration. J. Neural Eng. 2010, 7 (4), 046003. (22) Sun, M.; Kingham, P. J.; Reid, A. J.; Armstrong, S. J.; Terenghi, G.; Downes, S. In vitro and in vivo testing of novel ultrathin PCL and PCL/PLA blend films as peripheral nerve conduit. J. Biomed. Mater. Res., Part A 2010, 93A (4), 1470−1481. (23) Daly, W. T.; Knight, A. M.; Wang, H.; de Boer, R.; Giusti, G.; Dadsetan, M.; Spinner, R. J.; Yaszemski, M. J.; Windebank, A. J. Comparison and characterization of multiple biomaterial conduits for peripheral nerve repair. Biomaterials 2013, 34 (34), 8630−8639. (24) Qiu, Y.; Lim, J. J.; Scott, L.; Adams, R. C.; Bui, H. T.; Temenoff, J. S. PEG-based hydrogels with tunable degradation characteristics to control delivery of marrow stromal cells for tendon overuse injuries. Acta Biomater. 2011, 7 (3), 959−966. (25) Liu, X.; Paulsen, A.; Giambini, H.; Guo, J.; Miller, A. L.; Lin, P.C.; Yaszemski, M. J.; Lu, L. A New Vertebral Body Replacement Strategy Using Expandable Polymeric Cages. Tissue Eng., Part A 2017, 23 (5−6), 223−232. (26) Dadsetan, M.; Knight, A. M.; Lu, L.; Windebank, A. J.; Yaszemski, M. J. Stimulation of neurite outgrowth using positively charged hydrogels. Biomaterials 2009, 30 (23), 3874−3881. (27) Shin, H.; Ruhe, P. Q.; Mikos, A. G.; Jansen, J. A. In vivo bone and soft tissue response to injectable, biodegradable oligo(poly(ethylene glycol) fumarate) hydrogels. Biomaterials 2003, 24 (19), 3201−3211. (28) Martino, S.; D’Angelo, F.; Armentano, I.; Kenny, J. M.; Orlacchio, A. Stem cell-biomaterial interactions for regenerative medicine. Biotechnol. Adv. 2012, 30 (1), 338−351. (29) Poltawski, L.; Watson, T. Bioelectricity and microcurrent therapy for tissue healing−a narrative review. Physical Therapy Reviews 2009, 14 (2), 104−114. (30) Makohliso, S. A.; Valentini, R. F.; Aebischer, P. Magnitude and polarity of a fluoroethylene propylene electret substrate charge influences neurite outgrowth in vitro. J. Biomed. Mater. Res. 1993, 27 (8), 1075−1085. (31) Dillon, G. P.; Yu, X.; Bellamkonda, R. V. The polarity and magnitude of ambient charge influences three-dimensional neurite extension from DRGs. J. Biomed. Mater. Res. 2000, 51 (3), 510−519. (32) Chen, B. K.; Knight, A. M.; Madigan, N. N.; Gross, L.; Dadsetan, M.; Nesbitt, J. J.; Rooney, G. E.; Currier, B. L.; Yaszemski, M. J.; Spinner, R. J.; Windebank, A. J. Comparison of polymer scaffolds in rat spinal cord: A step toward quantitative assessment of combinatorial approaches to spinal cord repair. Biomaterials 2011, 32 (32), 8077−8086. (33) Hakim, J. S.; Esmaeili Rad, M.; Grahn, P. J.; Chen, B. K.; Knight, A. M.; Schmeichel, A. M.; Isaq, N. A.; Dadsetan, M.; Yaszemski, M. J.; Windebank, A. J. Positively Charged Oligo[Poly(Ethylene Glycol) Fumarate] Scaffold Implantation Results in a Permissive Lesion Environment after Spinal Cord Injury in Rat. Tissue Eng., Part A 2015, 21 (13−14), 2099−2114. (34) Madigan, N. N.; Chen, B. K.; Hakim, J. S.; Schmeichel, A. M.; Knight, A. M.; Zhang, S.; Nesbitt, J. J.; Dadsetan, M.; Chiang, T.; Yaszemski, M. J. Gdnf-secreting Schwann Cells in Multichannel Opf+

REFERENCES

(1) Pabari, A.; Yang, S. Y.; Seifalian, A. M.; Mosahebi, A. Modern surgical management of peripheral nerve gap. J. Plast Reconstr Aesthet Surg 2010, 63 (12), 1941−1948. (2) Alluin, O.; Wittmann, C.; Marqueste, T.; Chabas, J. F.; Garcia, S.; Lavaut, M. N.; Guinard, D.; Feron, F.; Decherchi, P. Functional recovery after peripheral nerve injury and implantation of a collagen guide. Biomaterials 2009, 30 (3), 363−373. (3) Belkas, J. S.; Shoichet, M. S.; Midha, R. Peripheral nerve regeneration through guidance tubes. Neurol. Res. 2004, 26 (2), 151− 160. (4) Trachtenberg, J. E.; Placone, J. K.; Smith, B. T.; Piard, C. M.; Santoro, M.; Scott, D. W.; Fisher, J. P.; Mikos, A. G. Extrusion-based 3D printing of poly (propylene fumarate) in a full-factorial design. ACS Biomater. Sci. Eng. 2016, 2 (10), 1771−1780. (5) Henry, M. G.; Cai, L.; Liu, X. F.; Zhang, L.; Dong, J. Y.; Chen, L.; Wang, Z. Q.; Wang, S. F. Roles of Hydroxyapatite Allocation and Microgroove Dimension in Promoting Preosteoblastic Cell Functions on Photocured Polymer Nanocomposites through Nuclear Distribution and Alignment. Langmuir 2015, 31 (9), 2851−2860. (6) Liu, X.; Miller, A. L.; Fundora, K. A.; Yaszemski, M. J.; Lu, L. Poly (ε-caprolactone) Dendrimer Cross-Linked via Metal-Free Click Chemistry: Injectable Hydrophobic Platform for Tissue Engineering. ACS Macro Lett. 2016, 5, 1261−1265. (7) Cai, L.; Foster, C. J.; Liu, X. F.; Wang, S. F. Enhanced bone cell functions on poly(epsilon-caprolactone) triacrylate networks grafted with polyhedral oligomeric silsesquioxane nanocages. Polymer 2014, 55 (16), 3836−3845. (8) Liu, X. F.; Chen, W. J.; Gustafson, C. T.; Miller, A. L.; Waletzki, B. E.; Yaszemski, M. J.; Lu, L. C. Tunable tissue scaffolds fabricated by in situ crosslink in phase separation system. RSC Adv. 2015, 5 (122), 100824−100833. (9) Fan, W. M.; Gu, J. H.; Hu, W.; Deng, A. D.; Ma, Y. M.; Liu, J.; Ding, F.; Gu, X. S. Repairing a 35-mm-long median nerve defect with a chitosan/PGA artificial nerve graft in the human: A case study. Microsurg 2008, 28 (4), 238−242. (10) Kalbermatten, D. F.; Kingham, P. J.; Mahay, D.; Mantovani, C.; Pettersson, J.; Raffoul, W.; Balcin, H.; Pierer, G.; Terenghi, G. Fibrin matrix for suspension of regenerative cells in an artificial nerve conduit. J. Plast Reconstr Aes 2008, 61 (6), 669−675. (11) Ding, F.; Wu, J. A.; Yang, Y. M.; Hu, W.; Zhu, Q.; Tang, X.; Liu, J.; Gu, X. S. Use of Tissue-Engineered Nerve Grafts Consisting of a Chitosan/Poly(lactic-co-glycolic acid)-Based Scaffold Included with Bone Marrow Mesenchymal Cells for Bridging 50-mm Dog Sciatic Nerve Gaps. Tissue Eng., Part A 2010, 16 (12), 3779−3790. (12) Pettersson, J.; Kalbermatten, D.; McGrath, A.; Novikova, L. N. Biodegradable fibrin conduit promotes long-term regeneration after peripheral nerve injury in adult rats. J. Plast Reconstr Aes 2010, 63 (11), 1893−1899. (13) Abu-Rub, M. T.; Billiar, K. L.; van Es, M. H.; Knight, A.; Rodriguez, B. J.; Zeugolis, D. I.; McMahon, S.; Windebank, A. J.; Pandit, A. Nano-textured self-assembled aligned collagen hydrogels promote directional neurite guidance and overcome inhibition by myelin associated glycoprotein. Soft Matter 2011, 7 (6), 2770−2781. (14) Khaing, Z. Z.; Schmidt, C. E. Advances in natural biomaterials for nerve tissue repair. Neurosci. Lett. 2012, 519 (2), 103−114. (15) Evans, G. R. D.; Brandt, K.; Katz, S.; Chauvin, P.; Otto, L.; Bogle, M.; Wang, B.; Meszlenyi, R. K.; Lu, L. C.; Mikos, A. G.; Patrick, C. W. Bioactive poly(L-lactic acid) conduits seeded with Schwann cells for peripheral nerve regeneration. Biomaterials 2002, 23 (3), 841−848. (16) Ngo, T. T. B.; Waggoner, P. J.; Romero, A. A.; Nelson, K. D.; Eberhart, R. C.; Smith, G. M. Poly(L-lactide) microfilaments enhance peripheral nerve regeneration across extended nerve lesions. J. Neurosci. Res. 2003, 72 (2), 227−238. (17) Uz, M.; Sharma, A. D.; Adhikari, P.; Sakaguchi, D. S.; Mallapragada, S. K. Development of multifunctional films for peripheral nerve regeneration. Acta Biomater. 2016, DOI: 10.1016/ j.actbio.2016.09.039. 14689


Research Article

ACS Applied Materials & Interfaces Hydrogel Scaffolds Promote Ascending Axonal Regeneration, Remyelination and Partial Locomotor Recovery Following Complete Spinal Cord Transection in Rats. Ann. Neurol. 2015, 78, S107. (35) Madigan, N. N.; Chen, B. K.; Knight, A. M.; Rooney, G. E.; Sweeney, E.; Kinnavane, L.; Yaszemski, M. J.; Dockery, P.; O’Brien, T.; McMahon, S. S. Comparison of cellular architecture, axonal growth, and blood vessel formation through cell-loaded polymer scaffolds in the transected rat spinal cord. Tissue Eng., Part A 2014, 20 (21−22), 2985−2997. (36) Rooney, G. E.; Knight, A. M.; Madigan, N. N.; Gross, L.; Chen, B.; Giraldo, C. V.; Seo, S.; Nesbitt, J. J.; Dadsetan, M.; Yaszemski, M. J. Sustained delivery of dibutyryl cyclic adenosine monophosphate to the transected spinal cord via oligo [(polyethylene glycol) fumarate] hydrogels. Tissue Eng., Part A 2011, 17 (9−10), 1287−1302. (37) McCaig, C. D.; Zhao, M. Physiological electrical fields modify cell behaviour. BioEssays 1997, 19 (9), 819−826. (38) McCaig, C. D.; Rajnicek, A. M.; Song, B.; Zhao, M. Controlling cell behavior electrically: current views and future potential. Physiol. Rev. 2005, 85 (3), 943−978. (39) Nuccitelli, R. Endogenous electric fields in embryos during development, regeneration and wound healing. Radiat. Prot. Dosim. 2003, 106 (4), 375−383. (40) Runge, M. B.; Dadsetan, M.; Baltrusaitis, J.; Ruesink, T.; Lu, L. C.; Windebank, A. J.; Yaszemski, M. J. Development of Electrically Conductive Oligo(polyethylene glycol) Fumarate-Polypyrrole Hydrogels for Nerve Regeneration. Biomacromolecules 2010, 11 (11), 2845− 2853. (41) Runge, M. B.; Dadsetan, M.; Baltrusaitis, J.; Knight, A. M.; Ruesink, T.; Lazcano, E. A.; Lu, L. C.; Windebank, A. J.; Yaszemski, M. J. The development of electrically conductive polycaprolactone fumarate-polypyrrole composite materials for nerve regeneration. Biomaterials 2010, 31 (23), 5916−5926. (42) Vivó, M.; Puigdemasa, A.; Casals, L.; Asensio, E.; Udina, E.; Navarro, X. Immediate electrical stimulation enhances regeneration and reinnervation and modulates spinal plastic changes after sciatic nerve injury and repair. Exp. Neurol. 2008, 211 (1), 180−193. (43) Behan, B. L.; DeWitt, D. G.; Bogdanowicz, D. R.; Koppes, A. N.; Bale, S. S.; Thompson, D. M. Single-walled carbon nanotubes alter Schwann cell behavior differentially within 2D and 3D environments. J. Biomed. Mater. Res., Part A 2011, 96 (1), 46−57. (44) Lee, J. H.; Lee, J.-Y.; Yang, S. H.; Lee, E.-J.; Kim, H.-W. Carbon nanotube−collagen three-dimensional culture of mesenchymal stem cells promotes expression of neural phenotypes and secretion of neurotrophic factors. Acta Biomater. 2014, 10 (10), 4425−4436. (45) Liu, X.; Miller, A. L., II; Park, S.; Waletzki, B. E.; Terzic, A.; Yaszemski, M. J.; Lu, L. Covalent crosslinking of graphene oxide and carbon nanotube into hydrogels enhances nerve cell responses. J. Mater. Chem. B 2016, 4 (43), 6930−6941. (46) Marcano, D. C.; Kosynkin, D. V.; Berlin, J. M.; Sinitskii, A.; Sun, Z.; Slesarev, A.; Alemany, L. B.; Lu, W.; Tour, J. M. Improved synthesis of graphene oxide. ACS Nano 2010, 4 (8), 4806−4814. (47) Dadsetan, M.; Szatkowski, J. P.; Yaszemski, M. J.; Lu, L. C. Characterization of photo-cross-linked oligo[poly(ethylene glycol) fumarate] hydrogels for cartilage tissue engineering. Biomacromolecules 2007, 8 (5), 1702−1709. (48) Dadsetan, M.; Hefferan, T. E.; Szatkowski, J. P.; Mishra, P. K.; Macura, S. I.; Lu, L.; Yaszemski, M. J. Effect of hydrogel porosity on marrow stromal cell phenotypic expression. Biomaterials 2008, 29 (14), 2193−2202. (49) Park, S.; Lim, B. B. C.; Perez-Terzic, C.; Mer, G.; Terzic, A. Interaction of asymmetric ABCC9-encoded nucleotide binding domains determines K-ATP channel SUR2A catalytic activity. J. Proteome Res. 2008, 7 (4), 1721−1728. (50) Park, S.; Hwang, I. W.; Makishima, Y.; Perales-Clemente, E.; Kato, T.; Niederlander, N. J.; Park, E. Y.; Terzic, A. Spot14/Mig12 heterocomplex sequesters polymerization and restrains catalytic function of human acetyl-CoA carboxylase 2. J. Mol. Recognit. 2013, 26 (12), 679−688.

(51) Pashley, R. M.; Rzechowicz, M.; Pashley, L. R.; Francis, M. J. De-gassed water is a better cleaning agent. J. Phys. Chem. B 2005, 109 (3), 1231−1238. (52) Peyton, S. R.; Raub, C. B.; Keschrumrus, V. P.; Putnam, A. J. The use of poly (ethylene glycol) hydrogels to investigate the impact of ECM chemistry and mechanics on smooth muscle cells. Biomaterials 2006, 27 (28), 4881−4893. (53) Xu, H.; Holzwarth, J. M.; Yan, Y.; Xu, P.; Zheng, H.; Yin, Y.; Li, S.; Ma, P. X. Conductive PPY/PDLLA conduit for peripheral nerve regeneration. Biomaterials 2014, 35 (1), 225−235. (54) Thrivikraman, G.; Lee, P. S.; Hess, R.; Haenchen, V.; Basu, B.; Scharnweber, D. Interplay of substrate conductivity, cellular microenvironment, and pulsatile electrical stimulation toward osteogenesis of human mesenchymal stem cells in vitro. ACS Appl. Mater. Interfaces 2015, 7 (41), 23015−23028. (55) You, J.-O.; Rafat, M.; Ye, G. J.; Auguste, D. T. Nanoengineering the heart: conductive scaffolds enhance connexin 43 expression. Nano Lett. 2011, 11 (9), 3643−3648. (56) Liau, B.; Zhang, D.; Bursac, N. Functional cardiac tissue engineering. Regener. Med. 2012, 7 (2), 187−206. (57) Shin, S. R.; Jung, S. M.; Zalabany, M.; Kim, K.; Zorlutuna, P.; Kim, S. B.; Nikkhah, M.; Khabiry, M.; Azize, M.; Kong, J.; Wan, K. T.; Palacios, T.; Dokmeci, M. R.; Bae, H.; Tang, X. W.; Khademhosseini, A. Carbon-Nanotube-Embedded Hydrogel Sheets for Engineering Cardiac Constructs and Bioactuators. ACS Nano 2013, 7 (3), 2369− 2380. (58) Roth, B. J. The electrical conductivity of tissues. In The Biomedical Engineering Handbook, 2nd ed.; CRC Press: 1999; 2 Vol. Set. (59) Ranck, J. B., Jr.; BeMent, S. L. The specific impedance of the dorsal columns of cat: an anisotropic medium. Exp. Neurol. 1965, 11 (4), 451−463. (60) Tasaki, I. A new measurement of action currents developed by single nodes of Ranvier. J. Neurophysiol. 1964, 27, 1199−1206. (61) Stout, D. A.; Basu, B.; Webster, T. J. Poly (lactic−co-glycolic acid): Carbon nanofiber composites for myocardial tissue engineering applications. Acta Biomater. 2011, 7 (8), 3101−3112. (62) Thrivikraman, G.; Mallik, P. K.; Basu, B. Substrate conductivity dependent modulation of cell proliferation and differentiation in vitro. Biomaterials 2013, 34 (29), 7073−7085. (63) Das, S. R.; Uz, M.; Ding, S.; Lentner, M. T.; Hondred, J. A.; Cargill, A. A.; Sakaguchi, D. S.; Mallapragada, S.; Claussen, J. C. Electrical Differentiation of Mesenchymal Stem Cells into SchwannCell-Like Phenotypes Using Inkjet-Printed Graphene Circuits. Adv. Healthcare Mater. 2017, 6, 1601087. (64) Li, L.; Davidovich, A. E.; Schloss, J. M.; Chippada, U.; Schloss, R. R.; Langrana, N. A.; Yarmush, M. L. Neural lineage differentiation of embryonic stem cells within alginate microbeads. Biomaterials 2011, 32 (20), 4489−4497.

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