SilkâGraphene Hybrid Hydrogels with Multiple Cues to Induce Nerve

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Bio-interactions and Biocompatibility

Silk-Graphene Hybrid Hydrogels with Multiple Cues to Induce Nerve Cell Behavior Lili Wang, Dawei Song, Xiaoyi Zhang, Zhaozhao Ding, Xiangdong Kong, Qiang Lu, and David L Kaplan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01481 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 18, 2018

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ACS Biomaterials Science & Engineering

Silk-Graphene Hybrid Hydrogels with Multiple Cues to Induce Nerve Cell Behavior Lili Wanga, Dawei Songb, Xiaoyi Zhanga, Zhaozhao Dinga, Xiangdong Kongc, Qiang Lua,*, David L. Kapland aNational

Engineering Laboratory for Modern Silk & Collaborative Innovation Center of Suzhou

Nano Science and Technology, Soochow University, Suzhou, Jiangsu, 215123, People’s Republic of China bTai’an

City Central Hospital, Taian, 271000, People’s Republic of China

cCollege

of Materials and Textiles, Zhejiang Sci-Tech University, Hangzhou, 310018, People’s

Republic of China dDepartment

of Biomedical Engineering, Tufts University, Medford, Massachusetts, 02155,

United States

Corresponding author: *Qiang

Lu, Tel: (+86)-512-67061649; E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT Cell behavior is dependent in part on chemical and physical cues from the extracellular matrix. Although the influence of various cues on cell behavior has been studied, challenges remain to incorporate multiple cues to matrix systems to optimize and control cell outcomes. Here, aligned silk fibroin (SF)-graphene hydrogels with preferable stiffness were developed through arranging SF nanofibers and SF-modified graphene sheets under an electric field. Different signals, such as bioactive graphene, nanofibrous structure, aligned topography, and mechanical stiffness were tailored into the hydrogel system, providing niches for nerve cell responses. The desired adhesion, proliferation, differentiation, extension and growth factor secretion of multiple nerverelated cells was achieved on these hydrogels, suggesting strong synergistic action through the combination of different cues. Based on the fabrication strategy, our present study provides a useful materials engineering platform for revealing cooperative influences of different signals on nerve cell behavior, to help in the understanding of cell-biomaterial interactions, with potential toward studies related to nerve regeneration.

KEYWORDS: silk, graphene, hydrogels, multiple cues, cell behavior


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1. INTRODUCTION Biomaterials used in regenerative medicine and tissue engineering should provide suitable microenvironments to actively control specific cellular behaviors and to stimulate functional regeneration of various tissues. Different physical and chemical properties, such as micro-nano topographies,1,2 stiffness,3,4 orientation,5,6 growth factors and chemical compositions provide effective cues to influence cell behavior and tissue regeneration.7-10 The synergistic influences of multiple cues exert stronger regulation of cell fate and have been extensively explored in recent years.11,12 However, introducing multiple tunable cues into the same biomaterial system remains challenge, with inherent technical barriers for the engineering of the biomaterials with this level of control. The formation of suitable niches containing various regulatory factors or cues is a key in biomedicine to foster continued improvements in cell and tissue outcomes.

Neuronal recovery remains a significant therapeutic challenge due to the high morbidity and serious permanent functional impairment of nerve damage worldwide, combined with the inferior regenerative capacity of nerves.13,14 Different strategies based on scaffolds and supportive tissues have been developed to create preferable microenvironments for stimulating neuronal regeneration. The fate and behavior of neuronal cells was guided by a variety of factors such as surface topography,15,16 orientation structure,17,18 stiffness/elasticity and conductivity of substrate,19-21 and external electrical and chemical stimulation.22-24 Many substrates with specific guiding cues have been optimized to improve nerve recovery, however, these substrates showed limited improvement towards regeneration.25-27 To improve on these outcomes, regulating multiple cues is needed, yet remains a challenge for the nerve tissue substrates. Thus,


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biomaterials that combine suitable 3D aligned nanofibrous structures, soft mechanical properties and conductivity would be useful towards nerve regeneration.

Silk fibroin (SF) biomaterials have been used as suitable matrices due to their satisfactory biocompatibility, tunable mechanical properties, and the formation of micro-nano hierarchical structures. Various neural cells including neural stem cells, neurons, schwann cells and gliocytes adhere, spread and proliferate on SF-based biomaterials, suggesting suitability of SF substrates towards nerve tissue regeneration.28-34 Different chemical and physical cues such as growth factors, nanofibrous structures, hydrogel state, and orientation were introduced to SF biomaterials to provide better microenvironments for nerve cells and tissues. Recently, SF biomaterials with multiple factors responsible for optimizing nerve regeneration were reported.35 For example, growth factor-loaded SF hydrogels with tunable stiffness were developed as soft matrices for neural tissue engineering.36,37 SF nanofibrous matrices with aligned structures were also prepared to guide the behavior of nerve cells.38 However, similar to other natural biomaterials, a challenge remains to form optimal niches with suitable cues for nerve regeneration.

Recent methods to assemble beta-sheet rich SF nanofibers in aqueous solutions, generating new SF scaffolds and hydrogels, have been successfully developed in our group.39 The nanofibers show suitable hydrophobic nature that allows sufficient entrapment and slow release of bioactive molecules without the loss of their biological functions. Further, electrical treatment generated nanofibrous hydrogels with aligned structures.40 Therefore, aligned SF nanofiber hydrogels are promising platforms for developing nerve tissue microenvironments with multiple cues. Here,


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graphene exfoliated with SF nanofibers as the stabilizer was incorporated into aligned SF hydrogels to generate bioactive matrices for nerve cells and to evaluate the feasibility of merging multiple cues on these platforms (Scheme 1).

Scheme 1. Schematic of SF-graphene hybrid hydrogels with multiple cues to induce nerve cell behavior.

The goal in present work was to combine multiple cues into SF scaffold systems to improve nerve cell outcomes. Thus, multiple controlling factors including nanofibrous structure, stiffness, alignment topography and electrical stimulation were successfully incorporated simultaneously into the materials and showed preferable and better nerve cell response.

2. EXPERIMENTAL SECTION 2.1 Preparation of SF Nanofiber Solutions. According to our prior methods, the SF concentrated solution (20 wt%) was diluted to 0.5 wt% and 1 wt% with ultrapure water, respectively, and then incubated at 60oC to induce the SF nanofiber solution.39 2.2 Preparation of Graphene Nanosheet Dispersions. The graphene nanosheet dispersions were obtained based on our recent SF nanofiber-exfoliating method.41 Simply, 200 mg of


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graphite powder was dispersed in 20 mL of 0.5 wt% SF nanofiber solution. The mixture was then sonicated 1 hour under 195 W and working pulse of 5s/2s in an ice-water bath. After standing for 24 hours, thick flakes were separated through centrifugation at 1,500 rpm for 30 minutes, obtaining graphene nanosheet dispersions with concentrations of about 2 mg mL−1.

2.3 Fabrication of Aligned SF-Graphene Hybrid Hydrogels. According to Table S1, the SFgraphene mixture was obtained by the introduction of various volumes of 2 mg mL−1 graphene nanosheet dispersions to 1 wt% SF nanofiber solutions. Then the volume of the mixture was adjusted to 16 mL with ultrapure water. Electrodes were immersed in the mixture and 50 V was applied. The graphene sheets were exfoliated with SF nanofiber as exfoliating agent and stabilizer.41,42 The obtained sheets were coated by SF nanofibers, endowing them similar negative charge. Therefore, the graphene sheets could migrate with the SF nanofibers under electrical fields to form aligned hybrid hydrogels. After 30 min, aligned composite hydrogels appeared near the positive electrode. The prepared samples were denoted ASH, 0.2G-ASH, 0.6G-ASH, 1.2G-ASH according to the graphene content. The 1wt% SF nanofiber hydrogel without electric field was also prepared as a control and termed RSH.

2.4 Hydrogels Characterization. Scanning Electron Microscopy (SEM). The freeze-dried hydrogels were sputter-coated with gold and then measured by SEM (Hitachi S-4800, Hitachi, Tokyo, Japan) at 3 kV. The microstructures of the hydrogels were analyzed by Image-Pro Plus software. At least 300 areas were randomly selected from each group. Finally, the alignment property of the hydrogels was qualified by FFT image analysis.40,42


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Transmission Electron Microscopy (TEM). The copper grid was immersed in the aligned SFgraphene hybrid hydrogels for 1-3 s and dried in air for 2 h. The distribution of graphene nanosheets in the hydrogel samples was viewed by TEM (Tecnai G2 F20 S-Twin, FEI Company, Hillsboro, USA) at 200 kV voltage.40

Swelling Ratio. The aligned SF-graphene hybrid hydrogels were swelled in ultrapure water for 24 h and weighed (Ws). Then these hydrogels were dried under vacuum and weighed (Wd). The swelling ratio was obtained according to the equation: Swelling ratio = (Ws - Wd)/Wd.42,43

Differential Scanning Calorimetry (DSC). The aligned SF-graphene hybrid hydrogels were freeze-dried and analyzed by DSC (TA Instruments, New Castle, DE, USA) at a heating rate of 2oC min-1 from -30 to 350oC to evaluate thermal properties.44

Mechanical Properties. The compressive properties of the aligned SF-graphene hybrid hydrogels were tested by a TMS-Pro Texture analyzer testing frame (TMS-PRO, FTC, USA).42 Hydrogels with dimensions of 4 mm diameter and 6 mm height were punched out from two different directions, i.e., parallel to and perpendicular to the SF layers, respectively. Six samples of each hydrogel were measured with the compression rate of 2 mm min-1. The compressive modulus was obtained from the stress-strain curve.


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Conductivity. The resistance (R) of the aligned SF-graphene hybrid hydrogels was measured using a multimeter (MS8226 digital intelligent multimeter, Extech, USA). The resistivity (ρ) was calculated with ρ= R A / L = R (W×H) / L, where A, L, H, and W represented the cross-sectional area, length, height, and width of the hydrogel sample, respectively. Conductivity (σ, S m −1) was calculated as the reciprocal value of resistivity, σ = 1 / ρ.45 Six samples of each hydrogel were measured.

2.5 Schwann Cells Culture and Proliferation. The rat schwann cells (SCs) were purchased from Shanghai Institutes for Biological Science and cultured in DMEM high glucose medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin. The different hydrogels were punched into round disks (height of 2 mm and diameter of 12 mm), fixed into 24-well plates and sterilized with 60Co γ-irradiation at 25 kGy. SCs were seeded on the hydrogels with cell density of 2.0×104 cells per well. After culturing for 1, 3 and 6 d, the samples were stained with Rhodamine-phalloidin and DAPI according to our previously reported protocols.42 CLSM (Confocal laser scanning microscope, Olympus FV10 inverted microscope, Nagano, Japan) was used to examine cell morphology on the hydrogels. At least 200 cells were measured using the Image J software to calculate the nuclear orientation angle. SCs proliferation was determined by DNA content assay on 1, 3 and 6 d.42 Simply, DNA of the cells was extracted using Omega's Tissue DNA kit, and then the fluorescence intensity (excitation wavelength 480 nm, emission wavelength 520 nm) was measured with a BioTeK spectrofluorometer (BioTeK, Winooski, VT, USA) to calculate the DNA content. Total RNA was extracted with Trizol reagent (Invitrogen, USA) and converted into cDNA. For detecting the mRNA level of BDNF (brain-derived neurotrophic factor), CNTF (ciliary neurotrophic factor) and NGF (nerve growth


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factor), the real-time qPCR was performed according to our previous report.46,47 GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control. The primer sequences used in SCs were given in Table S2.48 Each type of the hydrogel was analyzed in triplicate.

2.6 PC12 Cells Proliferation and Differentiation. PC12 cells were purchased from Shanghai Institutes for Biological Science, cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin and seeded on the hydrogels with a density of 3.0×104 cells per well. After culturing for 1, 3 and 6 d, PC12 cells proliferation was determined by DNA content assay.42

After culturing in 10% FBS medium supplemented with 50 ng mL-1 NGF for 6 d, the differentiation behavior of the PC12 cells was evaluated. PC12 cells were stained with FITCphalloidin and DAPI and visualized with CLSM for observation of cellular morphological features and neurite development. At least 200 cells were measured using Image J software to calculate the nuclear orientation angle. The neurite length of the cells and the ratio of PC12 cells with neurites were calculated.49,50 Above 300 PC12 cells were measured for each hydrogel. The mRNA expression levels of GAP43 (growth associated protein 43) and SYP (synaptophysin) in PC12 cells were measured by the real-time qPCR with GAPDH as an internal control.46,47 The primer sequences used in PC12 cells were given in Table S3.51 Each type of the hydrogels was analyzed in triplicate.


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2.7 Embryonic Stem Cells (ESCs) Differentiation. Mouse Embryonic stem cells (ESCs) were purchased from Shanghai Institutes for Biological Science. After the standard culture process,52,53 ESCs with 2.0×104 cells were seeded in each well. Differentiation medium (DMEM high glucose medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 0.1 mM β-mercaptoethanol, 5 µg/mL plasmocin prophlactic, 0.1 mM non-essential amino acids and 2.0 mM GlutaMAX) was replaced every other day.

Immunofluorescence staining of ESCs with β3-tubulin was used to characterize the differentiation of ESCs. Simply, after the standard washing, fixing, permeabilization, and blocking processes,52,53 the samples were stained in 1% BSA containing anti-β3-tubulin primary antibody overnight at 4oC and then future stained with goat anti-rabbit IgG secondary antibody (Abcam, USA) for 60 min.52,53 Finally, the nuclei of cells were stained with DAPI for 5 min. Representative immunofluorescence images were obtained by CLSM. With β-Actin as an internal control, the mRNA expression levels of Nestin and β3-tubulin in ESCs were analyzed by the real-time qPCR.46,47 The primer sequences used in ESCs were given in Table S4.52,53 Each type of hydrogel was measured in triplicate.

2.8 Statistical Analysis. SPSS software was applied in statistical analyses. One-way AVOVA was used to analyze the mean values of the data. P < 0.05 was considered statistically significant.

3. RESULTS AND DISCUSSION 3.1 Characterization of Aligned Silk-Graphene Hybrid Hydrogels. Soft biomaterials with 3D aligned micro-nanofibrous hierarchical structures are considered as suitable matrices for nerve


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regeneration.6,18,20 Silk nanofiber hydrogels have been used to culture nerve stem cells in vitro and induced the differentiation into neurons through tuning the stiffness.36,37 Aligned silk nanofiber hdyrogels were prepared under electrical fields and significantly influenced cell behavior, such as orientation and migration.42 However, the aligned hydrogels failed to actively induce the neural differentiation of stem cells and further modification is required to build suitable microenvironments for nerve regeneration. Graphene materials are considered promising bioactive nerve support matrices due to their electrical properties and biocompatibility.22,23,45,54 Recently, SF nanofibers as exfoliating agents were used to generate graphene nanosheets to form stable aqueous dispersions.41 Considering the natural compatibility of SF nanofibers and SF nanofiber-exfoliated graphene sheets, graphene sheets were introduced into the aligned hydrogels to achieve better niches for nerve regeneration.

Based on a preliminary study in our group,42 aligned hydrogels composed of 1 wt% SF nanofibers were chosen as a platform for the present study due to their suitable stiffness for nerve tissues. Various ratios of graphene dispersions were directly blended with the SF nanofiber aqueous solutions (Figure 1A and B) and then formed hydrogels under electrical fields. According to the graphene content (Table S1), the prepared samples were termed ASH, 0.2GASH, 0.6G-ASH, 1.2G-ASH, and the 1 wt% SF nanofiber hydrogel without electric field was used as a control and termed RSH. After these treatments, dark hydrogels appeared near the positive electrode while the remaining solution near the negative electrode became nearly transparent (Figure 1C). The results indicated that both SF nanofibers and graphene moved toward the positive electrode and formed composite hydrogels. Higher ratios of graphene resulted in darker hydrogels, suggesting an increase of graphene inside the hydrogels (Figure


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1D). Although the kinetics of the freezing process such as the quench rate could significantly change the microstructure of the hydrogels, our recent study suggested that the aligned SF nanofiber hydrogels could maintain their native microstructure after the lyophilization in liquid nitrogen.40 Therefore, the freeze-dried hydrogels in liquid nitrogen were measured with scanning electron microscopy (SEM) to reveal microstructural changes after the introduction of the graphene (Figure 2A). The shape and height of the FFT (Fast Fourier Transformation) curves indicated the alignment degree of the hydrogels. Two strong peaks existed for the aligned hydrogels, confirming the ordered orientation of the features (Figure 2B). Since the graphene sheets were coated by the SF nanofibers in the exfoliating process to improve the dispersion in water, the sheets exhibited superior compatibility with the SF nanofibers. TEM images of the layers further revealed that the graphene sheets were homogeneously dispersed in the SF nanofibers without aggregation (Figure 2C). Compared to pure SF nanofiber hydrogels, similar aligned lamellae structures with thickness of 4.3~4.4 µm and inter-lamellar distances of 24.3~25.1 µm appeared for these composite hydrogels (Figure 2D, E). The micro-structural features confirmed the successful introduction of hierarchical anisotropic cues, including aligned layers at micrometer scale and aligned nanofiers on the layers.


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Figure 1. Fabrication of aligned SF-graphene hybrid hydrogels: (A, B) Photograph and SEM image of SF nanofiber and SF nanofiber-graphene composite solutions; (C) Photographs of the phase changes before and after electrical field treatment; (D) Photographs of the aligned SFgraphene hybrid hydrogels.

Figure 2. The microscopic structures of the aligned SF-graphene hybrid hydrogels: (A) SEM images, (B) FFT image analysis, (C) TEM images, (D) Space width and (E) Lamella thickness of the aligned SF-graphene hybrid hydrogels. The inserts (A) are surface morphologies of the porous walls. The red arrows indicated the graphene dispersed in the aligned hybrid hydrogels.


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Unlike graphene nanosheets reported by other groups,55-57 the SF-exfoliated graphene materials were coated by SF nanofibers, endowing them with improved dispersibility in aqueous solutions and interactions with the SF nanofibers.41 High swelling ratios great than 11 were achieved for all the hydrogels, suggesting their good swelling capacity (Figure 3A). The swelling ratios decreased gradually from 18.7±1.5 to 16.2±1.2, 13.6±1.5 and 11.8±1.2 for ASH, 0.2G-ASH, 0.6G-ASH and 1.2G-ASH hydrogels, respectively, due to the hydrophobic property of graphene sheets. DSC curves exhibited degradation peaks at higher temperatures for the composite hydrogels with higher graphene contents, confirming the interactions between graphene and SF nanofibers (Figure 3B). Besides the influence of the anisotropic features, the introduction of graphene sheets could be used to tune the mechanical properties (Figure 3C), which was similar to our recent study where the graphene sheets could be introduced to silk films to significantly improve the mechanical property.41 Gradual anisotropic mechanical properties were achieved for these composite hydrogels where the compressive modulus increased from 0.4 kPa to 0.8 kPa when compressing the hydrogels orthogonal to the silk layers, while the values increased from 0.2 kPa to 0.4 kPa simultaneously when compressing parallel to the layers. The results suggested that suitable mechanical cues were achieved for these aligned hydrogels related to nerve tissue needs. Besides the mechanical properties, the inclusion of the graphene sheets also increased the electrical conductivity of the hydrogels, which should facilitate the neural differentiation of stem cells (Figure 3D). Therefore, as expected, SF nanofiber hydrogels with multiple regulating cues including aligned topography, nanofibrous structure, stiffness, and bioacitive graphene sheets were prepared through the introduction of graphene sheets, to provide active control of nerve cells.


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Figure 3. Characterization of the aligned SF-graphene hybrid hydrogels: (A) Swelling ratios, (B) DSC curves, (C) Compressive modulus and (D) Conductivity of the aligned SF-graphene hybrid hydrogels. Data represents mean±SD (n = 6), where *p < 0.05.

3.2 Cell Behavior on the Silk-Graphene Hybrid Hydrogels. To examine the effect of these multiple biomaterial cues on nerve cells, schwann cells (SCs) were seeded on the composite aligned hydrogels and pure SF nanofiber hydrogels with and without aligned structures. When nerve tissue damage happens, SCs, critical support cells in nerve tissue, migrate to the injury site and then proliferate and elongate to facilitate axon growth and myelinization.58,59 Meanwhile, the SCs also produce different growth factors to stimulate the regeneration of axons.60,61 In our in


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vitro cell culture, cell numbers increased after 6 days on all the SF nanofiber hydrogels, suggesting cytocompatibility (Figure 4A-C). Better cell proliferation appeared on the aligned hydrogels and further improved following the increase of graphene content, indicating active cues in optimizing the cyto-responses. The aligned features of the hydrogels exhibited a strong effect on cell morphology and directional growth for SCs. The cells showed a spread morphology and multidirectional growth on the homogeneous SF nanofiber hydrogels, and displayed similar elongated shapes and grew along the oriented layers on all the aligned SF nanofiber hydrogels with and without graphene sheets (Figure 4A, B). The angular distribution of cell elongation on the aligned hydrogels was about 30°, which was significantly narrower than that on the homogeneous hydrogels (Figure 4B).

Three important factors for nerve regeneration including BDNF (brain-derived neurotrophic factor), CNTF (ciliary neurotrophic factor) and NGF (nerve growth factor) were measured in the SCs-hydrogel system (Figure 4D-F). Similar to previous studies, significantly higher amounts of the factors secreted for the cells seeded on the aligned hydrogels, confirming the stimulating effect of aligned structure on SCs.62 The production of these factors was further improved after the introduction and increase of graphene sheets, which suggested stronger control through the multiple cues.


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Figure 4. The cell behavior of SCs on the hydrogels: (A) Confocal microscopy images of SCs cultured on day 1, 3, and 6. The blue color (DAPI) represents cell nucleus, while the red color (TRITC labeled phalloidin) represents actin cytoskeleton; (B) Distribution of SCs nucleus orientation angles when the cells seeded on the hydrogels were cultured for 6 days. The 90o angle means the angle perpendicular to the aligned direction of SF nanofiber layers; (C) SCs proliferation on these hydrogels measured with DNA analysis; and Relative gene expression of (D) BDNF, (E) CNTF and (F) NGF on these hydrogels. * Statistically significant P < 0.05.


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Rat pheochromocytoma 12 (PC12) cells were used to replace primary neuronal cells with in vitro models due to the similar neuronal properties, such as neurotransmitter synthesis and morphological differentiation.54,63 Similar to the SCs, the proliferation of PC12 cells improved following the introduction of aligned cues and was further optimized after the addition and content increase of graphene sheets (Figure 5A). Compared with the homogeneous SF nanofiber hydrogels, elongated and orientated morphologies along with the aligned layers were observed for the PC12 cells on the aligned hydrogels (Figure 5B). More oriented and longer neurites formed on the hydrogels with higher graphene content. The angular distribution of cell elongation on these aligned hydrogels was again about 30̊, significantly narrower than that on the homogeneous hydrogels (Figure 5C). Neurite length and percentage of neurite-bearing PC12 cells were counted to evaluate the level of neurite differentiation (Figure 5D, E). Both the neurite length and percentage of the neurite-bearing PC12 cells slightly increased on the aligned, pure SF nanofiber hydrogels and then significantly improved following the addition and content increase of graphene sheets. These results indicated that the graphene sheets effectively induced neurite differentiation, consistent with several previous studies.64-66 Different neurite-related gene expression markers, including GAP43 (growth associated protein 43) and SYP (synaptophysin) were measured to assess differentiation of the PC12 cells (Figure 5F, G). Consistently higher gene expression levels happened for the cells cultured on the aligned composite hydrogels, confirming the induction capacity of the composite hydrogels with more graphene sheet content to promote PC12 cell differentiation and neurite growth. Therefore, the PC12 cell culture results also indicated that multiple cues, such as orientation, graphene and


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hydrogel state provided effective synergy to improve the microenvironment for nerve regeneration.

Figure 5. The cell behavior of PC12 cells on the hydrogels: (A) PC12 cell proliferation on the hydrogels measured with DNA analysis; (B) Confocal microscopy images of PC12 cells cultured on the hydrogels for 6 days; (C) Distribution of nucleus orientation angles of PC12 cells cultured on the hydrogels for 6 days. The blue color (DAPI) displays cell nucleus, while the green color


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(FITC labeled phalloidin) represents actin cytoskeleton. The 90o angle indicates the angle perpendicular to the aligned direction of SF nanofiber layers; (D) Average neurite length of PC12 cells cultured on the hydrogels for 6 days; (E) Percentages of neurite-bearing PC12 cells cultured on the hydrogels for 6 days; and Relative gene expression of (F) GAP43 and (G) SYP of PC12 cells cultured on these hydrogels. *Statistically significant P < 0.05.

Figure 6. The differentiation behavior of ESCs on the hydrogels: (A) Immunofluorescent staining images of ESCs cultivated for 7, 14, 21 and 28 days on the different hydrogels. The blue color displays cell nucleus, while the green color represents β3-Tubulin, and Relative gene


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expression of (B) Nestin and (C) β3-Tubulin from ESCs cultured on different hydrogels. *Statistically significant P < 0.05.

To further reveal the action of multiple cues on nerve cell behavior, the function of mouse embryonic stem cells (ESCs) on the hydrogels was also studied. ESCs had the capacity to differentiate into all cell types including nerve cells in vitro and in vivo, thus providing options for nerve repair.52,53 The immune-fluorescence staining of β3-tubulin was used to evaluate the neuronal differentiation of ESCs at 7, 14, 21, and 28 days on the various hydrogels. Representative images were shown in Figure 6A. More neuron-like cells presented on the 1.2GASH hydrogels, indicating that the graphene indeed had a stimulation capacity on neural differentiation. The mRNA levels of Nestin and β3-tubulin (neural marker genes expressed at the early and late stages, respectively) were significantly higher in the ESCs cultured on the aligned composite hydrogels, confirming better inducing capacity of the composite hydrogels with more graphene sheets to promote ESCs differentiation (Figure 6B, C). These results were consistent with the immunostaining results.

4. CONCLUSIONS Aligned silk-graphene hybrid hydrogels were prepared in electric fields where graphene sheets were homogeneously dispersed in the hydrogels. Multiple cues including nanofibrous structure, aligned topography, stiffness, bioactive graphene sheets and hydorgel state were successfully introduced into SF-based materials, resulting in synergistic effects on nerve cell behavior. These new matrices for nerve tissue regeneration provide additional options for fundamental and


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applied studies related to neural regeneration, and the hydrogels developed here provide a useful platform for the study of cooperative effects of multiple cues on cell and tissue behavior.

SUPPORTING INFORMATION The amount and ratio of SF nanofiber and graphene solutions in preparing different composite hydrogels, PCR primer sequences used in SCs, PC12 cells and ESCs were supplied. This material is available free of charge.

ACKNOWLEDGMENTS The authors thank the National Key R&D Program of China (2016YFE0204400), and the NIH (R01NS094218, R01AR070975). We also thank the Social Development Program of Jiangsu Province (BE2018626) for support of this work.


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For Table of Contents Use Only Silk-Graphene Hybrid Hydrogels with Multiple Cues to Induce Nerve Cell Behavior Lili Wanga, Dawei Songb, Xiaoyi Zhanga, Zhaozhao Dinga, Xiangdong Kongc, Qiang Lua,*, David L. Kapland


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SilkâGraphene Hybrid Hydrogels with Multiple Cues to Induce Nerve

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