Collagen Nanofibers Facilitated Presynaptic ... - ACS Publications

Jun 23, 2014 - Neural stem cells (NSCs) are deemed to be a potential cell therapy for brain and spinal cord reconstruction and regeneration following ...
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Collagen Nanofibers Facilitated Presynaptic Maturation in Differentiated Neurons from Spinal-Cord-Derived Neural Stem Cells through MAPK/ERK1/2-Synapsin I Signaling Pathway Yanling Yin,† Peng Huang,‡,§ Zhu Han,‡ Guojun Wei,‡ Changwei Zhou,‡ Jian Wen,‡ Bo Su,‡ Xiaoqin Wang,*,∥ and Yansong Wang*,‡ †

Department of Neurobiology and Beijing Institute for Brain Disorders, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, PR China ‡ Department of Spine Surgery, Second Affiliated Hospital of Harbin Medical University, Haerbin, Hei Long Jiang 150086, PR China § Department of Spine Surgery, China Orthopaedics Department, Pla General Hospital Beijing 100083, PR China ∥ National Engineering Laboratory for Modern Silk, Soochow University, Suzhou, 215123, PR China ABSTRACT: Neural stem cells (NSCs) are deemed to be a potential cell therapy for brain and spinal cord reconstruction and regeneration following injury. In this study, we investigated the role of nanofibrous scaffolds on NSCs-derived neurons in the formation of neural networks. Miniature excitatory postsynaptic currents (mEPSCs) were recorded using the whole-cell patch clamp recording method after the spinal cord-derived NSCs were differentiated into neurons and cultured in vitro for 10−14 days. It was observed that the frequency of mEPSCs in the differentiated neurons cultured on both randomly oriented and aligned collagen nanofibrous scaffolds was higher than that on the collagen-coated control and can be inhibited by an ERK inhibitor (PD98059), indicating that the collagen nanofibers affected the maturation of the synapses from presynaptic sites via the MAPK/ERK1/2 pathway. In addition, both of the collagen nanofibers increased the phosphorylation of Synapsin I and facilitated the interaction of p-ERK1/2 and p-Synapsin I. All these results suggested that the collagen nanofibrous scaffolds contributed to the presynaptic maturation via the ERK1/2-Synapsin I signaling pathway.

1. INTRODUCTION The reconstruction and regeneration of the brain and spinal cord following injury is a formidable task that scientists have been performing for decades. Neural stem cells (NSCs) are multipotent cells with the ability to self-renew and differentiate into mature ones, such as neurons, astrocytes, and oligodendrocytes.1 Cell replacement therapy using transplanted NSCs is a promising technique, which contributes to various levels of functional recovery in animals after an experimental injury of the brain or spinal cord. Nevertheless, the tissue damage and loss after a CNS injury limit the survival and integration of transplanted NSCs. It remains challenging to develop new strategies for the maturation of the differentiated neurons, which may act as a promising target for the spinal cord injury. In response, researchers have developed many biomaterial substrates that have been used to culture, transplant, and influence the differentiation and integration of transplanted NSCs.2 The maturation of neural circuits relies on spontaneous electrical activity that occurs during immature stages of development.3 Physiological studies in various parts of the CNS indicate that during early development there are many © 2014 American Chemical Society

glutamatergic synapses where transmission is mediated by the activation of N-methyl-D-aspartic acid receptors (NMDARs) alone.4−6 NMDARs also play a role in spinal cord development. Synaptic NMDARs activation may help regulate dendritic outgrowth and establish final synaptic connections in developing rat dorsal horns. The synaptic function of the differentiated neurons is essential for rebuilding structures that can bridge the injury gap and enable signal connection. In previous studies, the physiological functions of the differentiated neurons were detected,1,7−9 and the effect of biomaterial substrates on the neural functions was addressed.10−12 However, little is known about the role of biomaterials in the synaptic maturation of differentiated neurons derived from stem cells. The extracellular matrix (ECM) is supposed to support the basal microenvironment for the stem cells. The basal interaction between ECM and stem cells depends on the characteristics of the topographic structures of ECM.13 The Received: March 1, 2014 Revised: June 12, 2014 Published: June 23, 2014 2449

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discarded and the cell pellet was resuspended in a complete neurobasal medium containing 1% glutamine, 3% fetal bovine serum, 1× B-27 supplement (all from Gibco), 5 ng/mL ciliary neurotrophic factor (CNTF), and 5 ng/mL brain-derived neurotrophic factor (BDNF) (Leinco). The cells were plated with a density of 2.5 × 104 per well onto poly(D-lysine) (Sigma) coated 8-well chamber slides (Labtek) and grown in a 37 °C incubator in a 5% CO2 environment. Unless otherwise specified, half of the culture volume was replaced with fresh medium every third day. 2.4. Flow Cytometry and Thiazolyl Blue Tetrazolium Bromide (MTT) Assay. To determine the cell viability, the differentiated cells were seeded at 1.0 × 105 cells in 24-well plates. Cellular death was measured after the treatment of cell suspension using propidium iodide (PI) (Sigma-Aldrich). The analysis was performed in a flow cytometer (BD FACSAria, Becton Dickinson, San Jose, CA). An MTT assay was also used to assess the cell viability. Ten microliters of MTT (Sigma-Aldrich, 5 mg stock in phosphate-buffered saline [PBS]) was added to each well (96-well plate, 100 μL of medium/well), and the plate was incubated for 4 h. The insoluble blue formazan was solubilized with 100 μL of dimethyl sulfoxide (DMSO), and the OD values of the mixture were measured at 550 and 650 nm with a Bio-Rad microplate reader. All the MTT assays involved no less than 4 separate samples, which were measured in triplicate. The survival of the cells cultured on the control was taken as 100%, with values for the other groups being given as a percentage of the control. 2.5. Immunofluorescence. After culturing, the cells were fixed with 4% buffered paraformaldehyde and stained using the previously described procedures.16 Representative fields from at least two independent experiments were imaged using a DFC300 digital camera and FW4000 imaging software (LEICA, Germany) and then scored. Then, 4′,6-diamidino-2-phenylindole (DAPI; Sigma) counterstaining of nuclei was used to determine the total number of cells in a field. The primary antibodies used for immunofluorescent detection included antibodies against phosphorylated-Synapsin I (1:200; Cell Signaling Technology, Inc. USA), phosphorylated-ERK1/2 (1:200; Cell Signaling Technology, Inc. USA), and glial fibrillary acidic protein (GFAP, 1:200; Sigma), monoclonal antibodies against microtubuleassociated protein 2a/b (MAP2a/b, 1:200; Chemicon) and galactocerebroside (GalC, 1:100; Sigma). The secondary antibodies used were Cy3-conjugated goat anti-mouse IgG (1:200, Sigma), and FITC-conjugated goat anti-rabbit IgG (1:400; Molecular Probe). The specificity of all the primary and secondary antibodies was confirmed in appropriate positive and negative control cultures. 2.6. Whole-Cell Patch Clamp Recording. The whole-cell voltage-clamp technique was used to record currents. The patch electrodes of thick-walled borosilicate glass (VWR Scientific, West Chester, PA) were pulled on a PP-83 micropipette puller (Narishige, Japan). The patch-pipet solution contained (in mM): 140 KCl, 10 Hepes, 10 EGTA, 2 MgCl2, 2 Na2ATP, 1 CaCl2, pH 7.3. The typical resistance of the glass electrodes was 3−5 MΩ when filled with intracellular pipet solution. Data were collected with an Axopatch 200B amplifier (Axon Instruments, Forster City, CA) and acquired and analyzed using pCLAMP 9.0 (Axon Instruments). The fast and slow capacitances were neutralized, and the series resistance was always compensated (approximately 70%). Miniature spontaneous excitatory postsynaptic currents (sEPSCs) were isolated by the application of the antagonist of the inhibitory γ-aminobutyric acid (GABAA) receptor, 50 μM bicuculline, the AMPA receptor antagonist DNQX (20 μM, Tocris) and 1 mM TTX (Sigma). mEPSCs were recorded without synaptic stimulation at a holding potential of −70 mV from differentiated neurons for at least 5 min. Neurons were selected for recording based on their morphology and the density of the surrounding cells. The criterion was that they were similar to primary cultured neurons. Relatively isolated or spherically shaped cells were avoided. 2.7. Sample Preparation and Western Blot Analysis. The cells were collected after differentiation on the control, randomly oriented, or aligned nanofibers for 10−14 days. The samples were dissociated in a lysis buffer containing 20 mM HEPES, pH 7.4, 1 mM EDTA, 1%

ECM may contribute to the stem cell adhesion, self-renewal, and differentiation. Besides, the structural characteristics of the ECM may play an important role in regulating the function and behavior of differentiated cells.14 Researchers lay more and more emphasis on electrospun nanofiber matrices because of their resemblance on the native ECM in intrinsic structures. In the present study, we synthesized collagen type I nanofibrous scaffolds with electrospinning. In our previous studies, it was observed that collagen nanofibers significantly promoted the proliferation of the stem cells.15 In addition, little is known about the role of collagen nanofibers on the maturation of synapses, which are of great importance for the reconstruction of neural circuits. To explore the effect of collagen nanofibers on the maturation of the synapses, we investigated the miniature spontaneous excitatory postsynaptic currents (mEPSCs) using the whole-cell patch clamp recording method and the phosphorylation of ERK1/2 and Synapsin I with a Western blot assay. Strikingly, it is determined that collagen type I nanofibrous scaffolds play an important role in the conformation of new synaptic contacts from the NSCs-derived neurons.

2. MATERIALS AND METHODS 2.1. Electrospinning. Randomly oriented and aligned collagen nanofibrous scaffolds were made as we previously reported.15 The ultrastructure patterns of the randomly oriented and aligned electrospun collagen fibers were evaluated by scanning electron microscopy (SEM, CamScan MX2600FE, UK). Finished samples were mounted on aluminum pegs and sputter coated with platinum nanoparticles for 1 min to reduce charging and produce a conductive surface. Tensile testing of the nanofibrous scaffolds was performed with a uniaxial mechanical tester (Instron 3365). Samples were excised into rectangular shape (40 × 5 mm2), and shapes of aligned samples were cut in the direction of alignment. No less than five samples per group were tested. In addition, the water adsorption capacity was calculated as before.15 2.2. Culture of Spinal-Cord-Derived NSCs. Neonatal Sprague− Dawley rats were purchased from the animal facility of the Second Affiliated Hospital of Harbin Medical University. All the experiments involving animals were approved by the Institutional Animal Care and Use Committee (IACUC). The NSCs were isolated from the neonatal spinal cord and cultured as previously described.12 The NSCs were passaged in a medium containing DMEM/F12 (Invitrogen, USA), NeuroCult NS-A proliferation supplement (Stem cell Technology, USA), 0.02% BSA (Sigma-Aldrich), 20 ng/mL epidermal growth factor (EGF) (Chemicon, USA), 10 ng/mL basic fibroblast growth factor (bFGF), and 0.002% heparin (Stem Cell Technology). After being sterilized under UV radiation for 2 h, the scaffolds were washed several times with phosphate buffered saline (PBS, pH 7.4) and soaked in DMEM/F12 for 24 h. The electrospun nanofibrous scaffolds were coated on the bottom of 96-well plates. Cell suspension in the proliferation medium was seeded at a density of 10 000 cells/well on the electrospun scaffolds. For the differentiation of the stem cells, the medium was changed to one containing DMEM/F12, 10% horse serum (Gibco Inc., Grand Island, NY, USA), and 1% glutamine (Invitrogen, USA). Half of the culture volume was replaced with fresh medium every third day until 10−14 days during the differentiation. 2.3. Primary Cell Culture of Spinal Cord. Spinal cords of neonatal rats (within 24 h) were carefully dissected under microscopy and were processed individually. The isolated spinal cords with meninges removed were cut into small pieces, incubated with 0.25% (w/v) Trypsin−EDTA (Gibco) for 15 min at 37 °C and mechanically dissociated. After trypsinization, an equal volume of equine serum (Gibco) was added, and the mixture was lightly triturated until a homogeneous cell suspension was achieved. The cell suspension was then transferred to the neurobasal medium containing 1% glutamate (Gibco) and centrifuged at 400g for 5 min. The supernatant was 2450

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Figure 1. SEM images and mechanical properties of collagen nanofibrous scaffolds. SEM image of aligned (A) and randomly oriented (B) fibers obtained at 5000× magnification. Scale bar: 5.0 μm. C and D show the swelling ratio and stress−strain curves of the collagen nanofibrous scaffolds, respectively. β-actin (band density of protein/band density of ERK1/2) was expressed as a percentage to that of the control group. For the detection of the phosphorylation level of ERK1/2, the ratio of pERK1/2 to ERK1/2 in the control group was recognized as 100%, and the values of the aligned or randomly oriented nanofibers groups were expressed as a percentage of that of the control group. 2.8. Coimmunoprecipitation. Next, 500 mg of total protein from the neural cells culturing on the control, randomly oriented, and aligned scaffolds were preabsorbed with 25 mL of protein A/GSepharose for 2 h at 4 °C. Immunoprecipitation was performed using 2 mg antibody for 6 h, and the samples were incubated with 30 mL of A/G-Sepharose for 12 h at 4 °C with constant rotation. For the control experiments, anti-p-ERK1/2 or anti p-Synapsin I antibody was replaced with the same volume of IgG. The immunoprecipitates were resolved in thiourea buffer (6 M urea, 2 M thiourea, 4% CHAPS) before the SDS−PAGE analysis. To reduce inter-individual variation, equal amounts of protein extracts from three individual chambers were pooled into one independent sample. Triplicates for each group were subjected to the gel analysis to obtain a statistical significance for protein differences. 2.9. Statistical Analysis. The data were expressed as the means ± SEM. One-way ANOVA and post hoc tests, as indicated in the Results section, were used to compare the differences (Sigmastat 3.04, Chicago, IL, USA). Statistical significance was taken at the level of p < 0.05.

Triton, and a protease inhibitor cocktail (Roche, USA) with a 31 gauge needle 10 times. The protein concentrations were determined using a micro BCA assay before Western blot analysis (Peirce, Germany). A total of 20 μg of proteins was loaded per lane. The protein bands were resolved by SDS-PAGE using 4−12% NUPAGE Bis-Tris gels (Invitrogen). The proteins were then transferred to a PVDF membrane (Millipore, USA) for 2 h at 100 V. After being blocked in 10% nonfat milk for 30 min, the membranes were probed with antip-Synapsin I antibody (1:1000, Cell Signaling Technology, Inc. USA) at 4 °C overnight. After 4 washes in PBST (0.1% Tween 20 in PBS), a horseradish-peroxidase-(HRP)-conjugated goat anti-rabbit IgG (1:10000) (Zymed Laboratories, USA) was applied for 1 h. Immunoblots were visualized using Super Signal West Pico chemiluminescent substrates (Pierce) and exposed to FUJI medical X-ray films (FUJIFILM Corporation, Japan). The membranes were then washed and stripped with a stripping buffer (Alpha Diagnostic Intl. Inc., USA) and then reprobed with anti-p-ERK1/2 (1:2000, Cell Signaling Technology, Inc.), anti-ERK1/2 (1:2000, Cell Signaling Technology, Inc.) and a β-actin antibody as the loading control. PD98059 and curcumin, which were the antagonist or agonist of ERK1/2, were purchased from Sigma-Aldrich (St. Louis, MO, USA). The quantitative analysis for immunoblotting was performed after scanning of the X-ray film with Quantitative-One software (Gel Doc 2000 imaging system, Bio-Rad Company, CA, USA). For the protein levels, the protein ratio of p-Synapsin I (band density of protein/band density of β-actin) was expressed as a percentage compared with that of the control group. For the detection of the phosphorylation level, the ratio of p-Synapsin I to β-actin in the control group was recognized as 100%, and the values of randomly oriented or aligned nanofibers groups were expressed as a percentage to the control group. For the analysis of the expression of ERK1/2, the protein ratio of ERK1/2 to

3. RESULTS 3.1. Morphology of Collagen Nanofibrous Scaffolds. SEM images showed specific characteristic ultrastructures with randomly oriented and aligned nanofibers, and no bead defect was observed for all samples. Figure 1A,B represented the 2451

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defects for all of the aligned and randomly oriented nanofibrous scaffolds.15 The morphology of the aligned and randomly oriented nanofibers and the insets showed the corresponding diameter distribution. In this study, the swelling ratios of the randomly oriented and aligned collagen nanofibers were 397.37% and 346.15%, respectively. Previously, we observed that the spinal cord-derived NSCs possessed the characteristics of neural stem cells on both the collagen nanofibers and the control, although the NSCs formed a greater number of spheres on the collagen nanofibers than on the collagen-coated control, which suggested that the proliferation of NSCs may be increased by the presence of collagen nanofibers.15 In the present study, we first tested the expression of the cytoskeletal proteins nestin and GFAP in NSCs on the collagen-coated control. Nestin and GFAP are the well-accepted markers for neural stem cells.17 In this study, they were used in the immunofluorescence assay to determine if the spinal-cord-derived cells maintained the properties of neural stem cells (Figure 2A,B,C). The results indicated that the cells did possess the properties of neural stem cells, forming the basis for further study. 3.3. Effect of Collagen Nanofibrous Scaffolds on the Cell Viability of Differentiated Cells from Spinal-CordDerived NSCs. An ideal biomaterial must provide an excellent milieu for cell survival, especially mechanical properties, such as elasticity. The effect of collagen nanofibrous scaffolds on the viability of differentiated cells was explored. It was observed that there was no significant difference between the experimental groups in terms of flow cytometry (Figure 3A,B,C,D) and MTT assays (Figure 3E). Compared with the control group, the collagen nanofibrous scaffolds did not have an effect on cell survival. The following studies were involved in the effect on the differentiation ratio and the cell functions. 3.4. Effect of Nanofibrous Scaffolds on the Differentiation Ratio of Spinal- Cord-Derived NSCs. We then analyzed the differentiation potential of spinal-cord-derived neural stem cells cultured on collagen nanofibrous scaffolds. We used microtubule-associated protein 2 (MAP2) to identify mature neurons (Figure 4A), galactocerebroside (GalC) to identify oligodendrocytes (Figure 4D), and GFAP to identify astrocytes (Figure 4G) on aligned collagen nanofibrous scaffolds. Ten to fourteen days after the withdrawal of serum, most cells had differentiated into neurons, oligodendrocytes, and astrocytes. The total number of cells was determined by the nuclei staining with DAPI (Figure 4B,E,F). These results demonstrated that spinal-cord-derived neural stem cells did possess the ability to differentiate into neural cells, which would facilitate the maintenance of neural functions. Random field counting of immunofluorescence images showed the number of

morphology of the scaffolds consisting of a uniform distribution of randomly oriented and aligned nanofibers. The diameters of them were 723 ± 235 nm and 601 ± 182 nm, respectively, likely due to the shearing effect caused by the rotating drum when the jet came into contact with the surface of the drum. Hydrophilicity of the tissue engineering scaffold plays a vital role in the fate of transplanted cells. The swelling ratios of collagen nanofibers were 397.37% (randomly oriented) and 346.15% (aligned), respectively (Figure 1C), determined in the same way as previously reported.15 There was a positive correlation between swelling ratio of the collagen nanofibers and the porosity in the fibers, so that they will absorb more water through capillary effect. Figure 1D showed the representative stress−strain curve of the randomly oriented and aligned collagen nanofibrous scaffolds. Corresponding to our results, the orientation of the nanofibers was reported to mainly contribute to mechanical properties of nanofibrous scaffolds. Table 1 reveals the strain properties of the control, randomly oriented, and aligned collagen fiber scaffolds. Table 1. Tensile Strengths of the Aligned (30 MPa) and Randomly Oriented Nanofibers (14.5 MPa) Were Higher than That of the Control Collagen (at 0.2 mm Thickness)

control randomly oriented aligned

elastic modulus (MPa)

tensile stress (MPa)

elongation at break (%)

6.36 ± 0.48 1.81 ± 0.32

0.61 ± 0.03 15.02 ± 3.16

9.59 ± 0.15 5.38 ± 3.06

1.91 ± 0.79

29.73 ± 3.52

6.12 ± 0.65

Certain features of electrospun nanofibrous scaffolds such as hydrophilicity, swelling ratio, and elasticity can be tailored to mimic the native microenvironment required for spinal cord engineering. Here, both randomly oriented and aligned scaffolds were highly porous with porosity over 70%. The highly porous and hydrated structure of a scaffold can promote spontaneous cell assembly into tissue architectures and maintain cellular functions.13 There is increasing evidence that the fibril microstructure of electrospun collagen type Ibased ECMs not only determines tissue-level mechanical properties but also provides instructive physicochemical features of the local cellular microenvironment. Cells sense and respond to the spatial distribution of fibrils mainly by forming cell−matrix adhesions through integrin-mediated binding of collagen adhesion domains.14 3.2. Spinal-Cord-Derived Cell Line Possesses the Potential Properties of Neural Stem Cells. As we have reported, the SEM result revealed that highly uniform and smooth nanofibers were formed without the occurrence of bead

Figure 2. Immortalized cell line possessing the properties of neural stem cells. Immunofluorescence showed the expression of nestin (A) and GFAP (B) in the immortalized cells under proliferative conditions, exhibiting the properties of neural stem cells. The data were obtained from triplicate experiments. Bar = 50 μm. 2452

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Figure 3. Nanofibrous scaffolds had no effect on the viability of the differentiated cells from NSCs. A, B, and C are the representative flow cytometry results. (D) Quantitative analysis of flow cytometry revealed that there was no significant difference between the experimental groups (p > 0.05, n = 6). (E) Quantitative analysis of the MTT assay revealed that there was no significant difference between the experimental groups (p > 0.05, n = 6). Data = mean ± SEM.

Figure 4. Collagen nanofibrous scaffolds had no effect on the differentiation ratio of NSCs. A, D, and G show the representative immunofluorescence images, which showed neurons (labeled by MAP2), oligodendrocytes (labeled by GalC), and astrocytes (labeled by GFAP), respectively. B, E, and F are the corresponding nuclei images. C, F, and I are the merge images. Bar = 50 μm.

differentiated astrocytes, oligodendrocytes, and neurons (Figure 4C,F,I). There was no significant difference in the differ-

entiation ratio of the cells on the collagen-coated control, randomly oriented, and aligned collagen nanofibrous scaffolds 2453

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members encoded by distinct genes (SYN1, SYN2, and SYN3) that in turn yield various splicing isoforms.24 Among these, Synapsin I is the best-characterized member. It has been reported that Synapsin I might function in regulating trafficking between the reserve pool and the readily releasable pool of synaptic vesicles through phosphorylation-dependent interactions with the actin cytoskeleton and the synaptic vesicle membrane in presynaptic terminals in mature neurons.25,26 The phosphorylation of Synapsin may result in its dissociation from synaptic vesicles in the processes activated by neuro-modulators and growth factors involved both in the modulation of neurotransmitter release and in the remodeling of growing neurons during development.27 It has been reported that the phosphorylation of Synapsin by MAPK/ERK1/2 kinase plays a critical role in the formation of synapses between Helix neurons in vitro.28 In addition, much evidence supports the role of MAPK/ERK1/2 kinase in the neurotrophic regulation of synapse formation and plasticity, suggesting that the MAPK/ERK1/2 pathway is of importance in synaptic maturation.29−31 In our previous reports, the phosphorylation of ERK1/2, the upstream kinase of Synapsin, was affected by collagen nanofibrous scaffolds.15 In this study, the expression level of ERK1/2 and the phosphorylation level of ERK1/2 and Synapsin I were detected using a Western blot assay (Figure 6A). The phosphorylation levels of ERK1/2 and Synapsin I in the differentiated cells on the randomly oriented and aligned scaffolds were higher than those in the control group (Figure 6B). When the inhibitor of p-ERK1/2 (PD98059, 10 μM) was added into the medium of neurons on collagen nanofibrous scaffolds, the phosphorylation level of ERK1/2 and Synapsin I decreased (Figure 6B,C). However, when curcumin (10 μM), the activator of MEK, which was the upstream of ERK1/2 and contributes to the phosphorylation of ERK1/2, was added into the medium of the neurons on the collagen-coated control, the phosphorylation level of ERK1/2 and Synapsin I increased (Figure 6B,C), indicating that ERK1/2 was responsible for the phosphorylation of Synapsin I. Because the phosphorylation of Synapsin I plays a role in the facilitation of the release of glutamate vesicles, the dephosphorylation of Synapsin I by PD98059 resulted in a decreased frequency of mEPSCs, which suggested that collagen-nanofibrous-scaffold-induced phosphorylation of Synapsin I contributed to the release of glutamate vesicles. Based on these results, it was concluded that collagen nanofibrous scaffolds promoted the maturation of the synapses via the ERK1/2-Synapsin I pathway. 3.7. Nanofibrous Scaffolds Are Associated with the Interaction between p-ERK1/2 and p-Synapsin I. Because it had been demonstrated that nanofibrous scaffolds contributed to the functional synapses, we hypothesized that the nanofibrous scaffolds might have also exerted an impact on the interaction between p-ERK1/2 and p-Synapsin I. As illustrated in Figure 7A, p-ERK1/2 and p-Synapsin I colocalized at the synapse in the differentiated neurons from the randomly oriented and aligned collagen nanofibrous scaffolds. In addition, the colocalizations were less prominent in the control group. To further confirm the effect of nanofibrous scaffolds, reciprocal coimmunoprecipitation (co-IP) was employed. As shown in Figure 7B, p-Synapsin I could reciprocally coimmunoprecipitate with p-ERK1/2 in the whole cell lysates from the differentiated cells cultured on both randomly oriented and aligned collagen nanofibrous scaffolds but not on the collagen-coated control. Both of these results indicated

(Table 2), suggesting that the collagen nanofibrous scaffolds did not affect the directional differentiation of the stem cells. Table 2. Differentiation Ratio of NSCs in the Different Experimental Groupsa neurons oligodendrocytes astrocytes a

control

randomly oriented

aligned

10.23 ± 0.49 27.66 ± 1.41 35.45 ± 1.40

9.58 ± 0.82 30.45 ± 1.75 33.47 ± 2.25

9.45 ± 0.50 25.78 ± 2.29 31.57 ± 3.48

Data are expressed as mean ± S.E.M., p > 0.05, n = 24.

3.5. Nanofibrous Scaffolds Facilitated the Formation of Functional Neural Network in the Differentiated Neurons. Glutamate is a major excitatory neurotransmitter in the CNS and plays an important role in functions such as synaptic plasticity, learning, and memory.18,19 Among these, NMDARs are essential for spinal cord development. Synaptic NMDAR activation may help regulate dendritic outgrowth and establish functional synaptic connections in the development of the CNS.20−22 To examine whether the differentiated neurons could form functional synapses, cells differentiated for 10−14 days that exhibited neuronal morphology were selected for electrophysiological recordings. To explore the effect of collagen nanofibrous scaffolds on the synaptic function, we recorded the mEPSCs mediated by NMDARs in the differentiated neurons. mEPSCs of the neurons may be mediated by action potential-independent spontaneous release of glutamate from presynaptic terminals.23 A significant difference was observed in the frequency of mEPSCs between the neurons on collagen nanofibrous scaffolds and those on the collagencoated control (Figure 5B,C,D,H), suggesting an increased presynaptic glutamate release. No significant difference in the frequency was detected between the randomly oriented and aligned group (Figure 5B,C,H), suggesting that both groups facilitate the maturation of synapses. Furthermore, there was no significant difference in the frequency of mEPSCs between the differentiated neurons on the collagen nanofibrous scaffolds and the primary cultured neurons from the spinal cord (Figure 5A,B,C,H), indicating that the collagen nanofibrous scaffolds were only involved in the maintenance of normal synaptic function without excitotoxicity. There was no significant difference in terms of the mean amplitude of mEPSCs between the experimental groups (Figure 4G), suggesting that the postsynaptic reactivity of NMDARs was similar. In our previous study, we observed that collagen nanofibrous scaffolds increased the phosphorylation of ERK1/2. To investigate whether ERK1/2 is involved in the maturation of synapses, PD98059, an inhibitor of ERK1/2, was added into the medium before the recording. It turned out that the inhibition of phosphorylation of ERK1/2 prevented collagen nanofibrousscaffold-induced presynaptic maturation (Figures 5E,F,H), indicating that the collagen nanofibrous scaffolds may affect the functions of the synapses through the ERK1/2 signaling pathway. 3.6. Effect of Nanofibrous Scaffolds on the Maintenance of Functional Synapses via the Presynaptic ERK1/2/Synapsin I Signaling Pathway. Although the data indicated that collagen nanofibrous scaffolds affect cell functions through presynaptic modulation, little is known about the mechanism involved. Synapsins are a family of neuronal phosphoproteins associated with the cytosolic surface of synaptic vesicles. In mammals the family comprises three 2454

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Figure 5. Collagen nanofibrous scaffolds affected the generation of functional neurons from spinal-cord-derived NSCs. Representative traces of mEPSCs from primary cultured neurons (A), cells cultured on the randomly oriented collagen nanofibrous scaffolds (randomly group) (B), cells cultured on the aligned collagen nanofibrous scaffolds (aligned group) (C), cells cultured on the collagen-coated control (D), cells cultured on randomly oriented collagen nanofibrous scaffolds with pretreatment of PD98059 (the inhibitor of p-ERK1/2) (E), and cells cultured on aligned collagen nanofibrous scaffolds with pretreatment of PD98059 (F). (G) and (H) Bar graphs of the mean amplitude and frequency of mEPSCs in primary cultured (n = 12), randomly oriented (n = 11), aligned (n = 11), control (n = 10), randomly oriented +PD98059 (n = 10), and aligned +PD98059 (n = 10) groups. There was a significant difference in the frequency of mEPSCs between the collagen-coated control and PD98059 pretreatment groups vs that of the primary cultured groups (*p < 0.05). There was a significant difference in the frequency of mEPSCs between the aligned+PD98059 and aligned groups ($p < 0.05) and between the randomly oriented +PD98059 and the randomly oriented group (#p < 0.05). No significant difference in the frequency was detected between randomly oriented and aligned group. There was no significant difference in the mean amplitude between the 6 groups of mEPSCs. The data were expressed as means ± SEM.

neural tissue engineering, because neural progenitors and progeny, such as most other mammalian cells, are anchoragedependent and require attachment to a solid surface.33,34 In addition, the biomaterial can also contribute to the function of transplanted cells.35 In the present study, collagen type I was selected because collagen is a biologically derived biomaterial and the major class of insoluble fibrous protein in the ECM. It has been demonstrated that neural stem cells isolated from embryonic rat CNS tissue rapidly proliferate and differentiate into neurons and astrocytes in collagen type I gels.36 The

that nanofibrous scaffolds were favorable for the p-ERK1/2/pSynapsin I interaction, which may be involved in the maintenance of synaptic functions.

4. DISCUSSION Regenerative medicine refers to a group of biomedical approaches to clinical therapies that may involve the use of stem cells, which may be a promising therapeutic tool for many disorders in the central nervous system.32 The biomaterial provides a supportive scaffold, which plays a critical role in 2455

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Figure 6. Collagen nanofibrous scaffolds facilitated the maturation of dendrites/axons via the ERK1/2-Synapsin I pathway. A. Representative results. B. The phosphorylation level of Synapsin I in neurons on collagen nanofibers (randomly oriented and aligned) is higher than that on the collagencoated control. The phosphorylation level of Synapsin I in neurons on collagen nanofibers can be reduced by PD98059, the inhibitor of p-ERK1/2, which suggested that the activation of ERK1/2 plays a vital role in phosphorylation level of Synapsin I maintained by collagen nanofibers. In addition, curcumin, the activator of MEK that can phosphorylate ERK1/2, increased the phosphorylation level of Synapsin I in the neurons on the collagen-coated control. C. The phosphorylation level of p-ERK1/2 in neurons on collagen nanofibers (randomly oriented and aligned) is higher than that on the collagen-coated control. The activation of ERK1/2 can be inhibited by PD98059 and activated by curcumin. These results indicated that collagen nanofibrous scaffolds facilitated the maturation of dendrites/axons via the ERK1/2-Synapsin I pathway. The data were expressed as means ± SEM.

collagen nanofibers, compared with those on collagen-coated surfaces.15 Currently, researchers emphasize the effect of scaffolding materials on the function of NSC-derived neurons. Nevertheless, little is known about the effect of materials on the maturation of synapses, which are of great importance in the maintenance of neural functions. In a recent report, Bi-Qin Lai et al. reported that a gelatin sponge scaffold helped the NSCderived neurons form new synaptic contacts, suggesting that the scaffold can form a relay for the conduction of signals

exploration of how the fate of the cell can be affected by the substrate properties will contribute to the rational design and therapy strategies based on stem cells. Recent developments in material science, engineering, biotechnology, and biomedical fields have clearly demonstrated the many potential applications of nanotechnology, which can induce a better cellular response than untreated surfaces after cells are placed in contact with these materials.37,38 In our previous study, an increase in the proliferation and an elevation of BrdU incorporation were observed to occur on NPCs cultured on 2456

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Figure 7. Collagen nanofibers were associated with the interaction between p-ERK1/2 and p-Synapsin I, which may be essential for the maintenance of synaptic functions. A. Representative immunofluorescence images (n = 4) of p-ERK1/2 (red), p-Synapsin I (green), and DAPI (blue) staining in differentiated neurons cultured on the collagen-coated control, randomly oriented and aligned collagen nanofibrous scaffolds. The merged images show the colocalization of p-ERK1/2 and p-Synapsin I at synapses. Bar = 25 μm. B. Representative results of reciprocal IP revealed that p-ERK1/2 and p-Synapsin I could be coimmunoprecipitated by each other from the samples of differentiated cells in the control, randomly oriented, and aligned groups. Input and IgG were used as positive and negative controls, respectively (n = 3).

through the injury gap of the spinal cord.39 Still, the effect of collagen nanofibers on the functional synapses between the NSC-derived neurons was obscure. In the current study, we investigated the effect of electrospun collagen type I nanofibrous scaffolds on the synaptic maturation between NSCderived neurons, and the possible mechanism. In the CNS, neural cells adhere to the fibrillar protein meshwork, which is known as the ECM. The ECM is a critical determinant of cell behavior and is known to affect intracellular signaling pathways, cell differentiation events, and cell proliferation, among other important characteristics of tissue identity.40,41 The ECM may provide a microenvironment, in which extrinsic signals such as ECM proteins and soluble factors from other cells control the proliferation and fate of stem cells.42 Thus, the proper design of scaffold matrices may allow them to mimic the native ECM and contain necessary extrinsic factors for generating neural tissues from stem cells in a system in vitro.43 It is increasingly apparent that the fibril microstructure of electrospun collagen type I-based ECMs not only determines the tissue-level mechanical properties, but also provides instructive physicochemical features of the local cellular microenvironment. Cells sense and respond to the spatial distribution of fibrils largely by forming cell−matrix

adhesions through integrin-mediated binding of collagen adhesion domains.44 To explore the effect of randomly oriented and aligned oriented nanofibers on the maturation of the synapses, mEPSCs were recorded using the whole-cell recording method. The decrease of the frequency of mEPSCs indicated that the randomly oriented and aligned nanofibers mainly facilitated the presynaptic maturation. Furthermore, the mean amplitude of the mEPSCs can reflect the reactivity of the postsynaptic receptors. In this study, both the oriented nanofibers had no significant effect on the amplitude of mEPSCs, suggesting that the nanofibers were important in the presynaptic but not postsynaptic maturation. To elucidate the mechanism of the nanofibers on the electrophysiological functions of differentiated neurons, the phosphorylation and expression of synapsin I were detected in our study. Synapsins are a family of vesicle-associated and neuron-specific phosphoproteins that are clearly involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis.45,46 Synapsins interact with synaptic vesicle (SV) proteins, phospholipids and actin, regulating SV homeostasis.45 In our study, it was observed that randomly oriented and aligned nanofibers 2457

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Figure 8. Possible scenario for collagen nanofibers contributing to the presynaptic maturation. The red arrow represents the possible regulation of βintegrin to the activation of ERK1/2 during the maintenance of synaptic function. The green arrows represent the process for β-integrin detecting the extracellular signal of collagen nanofibers and p-ERK1/2 activating Synapsin I, which may play a role in the transportation of synaptic vesicles (SV) with actin.

the proliferation, differentiation, and migration of stem cells.56 The β-integrin-dependent signaling pathway may activate an ERK signaling cascade mediated by tyrosine kinase.20 In our previous study, we observed that collagen nanofibers play a role in the proliferation of stem cells via the β-integrins-ERK1/2 signaling pathway.15 In summary, we propose our hypothesis that collagen nanofibers are beneficial for the maturation of differentiated neurons via the β-integrins-ERK1/2-Synapsin pathway (see Figure 8). Collagen-based biomaterials have been widely used for tissue engineering. Collagen I is widely available from bovine, porcine, or, more recently, recombinant sources.57 The goal of biochemical modifications is to immobilize biologically active molecules on nanofibrous scaffolds surfaces to induce specific cell and tissue responses.58 The existence of collagen on the surface provides uninterrupted cell recognition signals, which is essential for cell function and development, including cellular attachment, survival, proliferation, differentiation, and the maintenance of normal functions in its native state.59 Certain features of electrospun nanofibrous scaffolds such as their hydrophilicity, swelling ratio, and elasticity can be tailored to mimic the native microenvironment required for spinal cord engineering. Here, both randomly oriented and aligned scaffolds were highly porous with porosities over 70%. The characteristic of hydrophilicity is important for tissue engineering scaffolds, which would affect the proliferation and maintenance of neuronal functions. The highly porous and hydrated structure of a scaffold may facilitate spontaneous assembly of cells into tissue architecture and maintain the cell routine life. In our previous study, we observed that the elastic modulus of collagen nanofibers is appropriate for self-renewal of spinal-cord-derived NSCs.15

increased the phosphorylation of Synapsin I. It has been reported the phosphorylation of Synapsin I, the bestcharacterized member of the family, is of great importance in both the maintenance of a depot of synaptic vesicles, thereby regulating SV functional pools and tuning neurotransmitter release,47−49 and the mobilization of vesicles from the reserve pool to the releasable pool under conditions of increased presynaptic activity.50−54 Therefore, aligned and randomly oriented nanofibers may induce presynaptic maturation through the phosphorylation of Synapsin I. The interaction of Synapsin I−actin may facilitate the release of synaptic vesicles. Synapsin is a substrate of several protein kinases, including PKA, CaMKs, and MAPK/ERK, which phosphorylate and modulate its biochemical properties. Carlo Natale et al. reported that MAPK/ERK-dependent phosphorylation of synapsin mediates the formation of functional synapses and short-term homosynaptic plasticity.55 In our previous study, we observed that nanofibers promoted the proliferation in spinalcord-derived neural stem cells through the MAPK/ERK1/2 signaling pathway.15 To investigate the role of MAPK/ERK1/2 in the differentiation of the neural stem cells, the phosphorylation and expression of ERK1/2 were detected using a Western blot assay. The results indicated that the nanofibers increased the phosphorylation of ERK1/2 compared with collagen, suggesting that collagen could not supply the required signaling for the synaptic maturation, while nanofibers could supply the excitoxic signal. The inhibition of ERK1/2 activation decreased the phosphorylation of Synapsin I, indicating that the nanofibers promoted presynaptic maturation via the MAPK/ ERK1/2 signaling pathway. In addition, the inhibition of ERK1/2 blocked the maintenance of mEPSCs due to nanofibers, confirming that nanofibers are involved in the ERK1/2 signaling pathway. It was observed that the nanofibers promoted the interaction between p-ERK1/2 and p-Synapsin I, which may be essential for the development of mature synapses. Integrins facilitate binding and the interactions of cells with components of the ECM in addition to cell−cell interaction. It has been reported that β-integrins transmit cell signals to help

5. CONCLUSIONS Porous scaffolds composed of collagen nanofibers were developed in the present study, which exhibited a large effect on the proliferation of neural stem cells and the synaptic formation of differentiated neurons. To measure the interactions between the collagen nanofibers and differentiated neurons, the differentiation ratio, synaptic functions, and so 2458

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forth were investigated in vitro. Potential mechanisms underlining the promoting effects of collagen nanofibers were addressed as collagen nanofiber scaffolds: (1) facilitated the maturation of synapses of differentiated neurons; (2) significantly activated ERK1/2, up-regulating the phosphorylation of ERK1/2; and (3) maintained the interaction between p-ERK1/2 and p-Synapsin I. It was also observed that collagen nanofibers facilitated the maintenance of synaptic functions, which may be the result of cross-talk between the biomaterials and the neurons dependent on the topography and the mechanical properties of the collagen nanofibers. Collectively, collagen nanofibers are beneficial for both the proliferation of stem cells and the maturation of differentiated neurons, thus providing a promising scaffold for using NSCs for treating spinal cord injuries.

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +86 451 8660 5599. Fax: +86 512 65883371. E-mail: [email protected]. *Phone: +86 451 8660 5599. Fax: +86-51 8660 5678. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the following grants: National Natural Science Foundation of China (31200895, 81271696), Beijing Municipal Talents Project (2013D005018000010), the Nature Science Foundation of Hei Long Jiang Province (ZD200916 and D200902), the WLD Foundation of Harbin Medical University (QN1115), and the Basic and Clinical Cooperation Project of the Capital Medical University (14JL77).

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REFERENCES

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