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The inhospitable niche at the injury site after spinal cord injury (SCI) brings several challenges to neural stem cell (NSC) therapy, such as limited NSC retention and neuronal differentiation. Biomaterial-based stem cell transplantation has become a promising strategy for building a favorable niche to stem cells. Herein, an aligned fibrin nanofiber hydrogel modified with N-Cadherin-Fc (AFGN) was fabricated by electrospinning and biochemical conjugation to deliver NSCs for SCI repair. The AFGN hydrogel provides multimodal cues, including oriented nanofibrous topography, soft stiffness, and specific cell binding ligand, for directing NSC functions and nerve regeneration. The conjugated N-Cadherin-Fc recapitulated the homo- philic cell–cell interaction for NSCs’ adhesion on AFGN and modulated cellular mechanosensing in response to AFGN for NSC differentiation. In addition, the AFGN hydrogel carrying exogenous NSCs was implanted in a rat 2 mm-long complete transected SCI model and significantly promoted the grafted NSCs retention, immunomodulation, neuronal differentiation, and in vivo integration with inherent neurons, thus finally achieved renascent neural relay formation and an encouraging locomotor functional recovery. Altogether, this study represents a valuable strategy for boosting NSC-based therapy in SCI regeneration by engineering an NSC-specific niche. Keywords N-cadherin · Aligned fibrin nanofiber scaffold · Niche · Neural stem cell · Spinal cord injury Introduction Spinal cord injury (SCI) is a devastating case leading to irreversible deficits in motor and sensory functions, pos- ing a worldwide threat to individuals and public health . Kaiyuan Yang, Jia Yang, and Weitao Man have contributed equally During the last decade, neural stem cell (NSC)-based tis- to the work. sue engineering therapies have been widely recognized as a promising tactic for nerve regeneration and lost function * Guihuai Wang reconstruction after SCI . Following transplantation into firstname.lastname@example.org injury sites, exogenous NSCs can differentiate into neural * Xiumei Wang cells and replenish the disastrous loss of neural cells after email@example.com SCI . Moreover, NSCs can act as a nanomaterial factory State Key Laboratory of New Ceramics and Fine that continuously provides various neurotrophic factors and Processing, Key Laboratory of Advanced Materials, School exosomes, which encourages axon re-extension and synapse of Materials Science and Engineering, Tsinghua University, reformation . However, due to the inhospitable niche at Beijing 100084, China the injury site after SCI, the low-grafted NSC retention and Department of Neurosurgery, Beijing Tsinghua Changgung neuronal differentiation extremely restrict the efficiency of Hospital, School of Clinical Medicine, Tsinghua University, Beijing 102218, China NSC transplantation therapy . Hence, reconstructing a desirable niche for NSCs has attracted much attention in School of Materials Science and Engineering, Zhejiang-Mauritius Joint Research Center for Biomaterials NSC-based SCI therapies [5–7]. and Tissue Engineering, Zhejiang Sci-Tech University, Hangzhou 310018, China Vol.:(0123456789) 1 3 Advanced Fiber Materials Recently, biomaterial scaffolds have not only served as advances in tissue engineering . N-Cadherin (NCAD), a platform to bridge the nerve gap after SCI but also been a type of cadherin that exists in the central nervous system actively used to engineer stem cell niche characteristics [8, (CNS), plays a crucial role in proper CNS development, neu- 9]. Biologically compatible materials, such as fibrin, colla- rite sprouting, and axon regeneration . NCAD is a homo- gen, polypeptides, and chitosan, could simulate the extracel- philic transmembrane glycoprotein that mediates cell–cell lular matrix (ECM), providing biophysical and biochemical adhesive contacts and modulates the cell–matrix mechani- cues to guide nerve regeneration [10–13], and have been cal response, which has been accepted as a vital determi- widely investigated for regulating stem cell behaviors and nant of stem cell fate [27–29]. The cytoplasmic regions of differentiation fate [14, 15]. For example, many studies have NCAD can form a cadherin–catenin complex by binding to indicated that scaffolds with aligned nanoscale topography α-catenin and β-catenin . The complex anchors actin mimicking the highly oriented structure of nerve fibers filaments (F-actin) that allow the cytoskeleton to sense and could better promote axon sprouting and affect cell fates respond to mechanical coupling across the cell membrane . Moreover, an increasing number of studies have shown modulate cell adhesion and migration [30, 31]. In addition, that the combination of different types of cues delivered by NCAD also encourages axon outgrowth and dendritic arbo- biomaterial scaffolds is more promising in cell delivery and rization through fibroblast growth factor receptor (FGFR) tissue regeneration than the single cue release strategy [6, activation [27, 32]. Notably, during the development of the 11]. Thus, in this study, we aimed to develop a novel bio- CNS, NCAD expressed on the surface of NSCs regulates functional scaffold that represents multimodal cues to better NSC adhesion and differentiation . Moreover, NCAD- reconstruct the NSC niche and guide axon regeneration. maintained adhesion could promote the differentiation of As a significant component of the ECM, fibrin offers NSCs toward neurons by regulating the Wnt/β-catenin and excellent biocompatibility, tunable degradability, suitable AKT signaling pathways [34, 35]. To better utilize and pre- flexibility, and plasticity [17, 18]. Moreover, fibrin contains sent NCAD in tissue engineering, recombinant N-cadherin many binding sites for ECM proteins as well as growth fac- human Fc chimeric protein (NCAD-Fc) was constructed tors and cell receptors, making fibrin a promising modifi - . NCAD-Fc contains the extracellular domain of NCAD able cell–carrier matrix and candidate for tissue engineering and the Fc domain of human immunoglobulin (IgG). The Fc . Over the past 10 years, various fibrin scaffolds have domain could enhance the activity, stability, and solubility demonstrated strong potential for SCI repair [3, 18, 20]. of NCAD . Furthermore, the Fc domain enables NCAD For example, a recent study reported that collagen-fibrin to immobilize onto substrates via hydrophobic interactions, hydrogel exhibits better mechanical properties and adhesive protein A (PA) affinity capture, or antigen–antibody inter - strength than single collagen hydrogel, and this composite actions [36, 38, 39]. Therefore, the NCAD-Fc-conjugated hydrogel accelerated endogenous NSC migration and SCI bioactive scaffold may be an attractive platform for NSC repair . In our previous study, an aligned fibrin nanofiber delivery, where NSCs can be retained in a desirable niche hydrogel (AFG) was constructed by electrospinning, which and differentiate toward neurons. Nevertheless, current stud- generated a nanoscale linearly ordered structure and soft ies mainly focused on NCAD-Fc as a cofactor for maintain- elasticity mimicking native nerve ECM. Furthermore, the ing cell adhesion cultures or promoting NSC neuronal dif- 3D nanoscale construction of AFG provides a high surface ferentiation on two-dimensional interfaces in vitro [26, 31, area for cell attachment and growth . We found that AFG 36, 40], and the potential of NCAD-Fc-conjugated bioactive could promote the neurogenic differentiation of mesenchy - scaffolds modulating the grafted NSC niche as a combinato- mal stem cells (MSCs) and activate the oriented neurite rial strategy to promote SCI repair is overlooked. project of rat dorsal root ganglion (DRG) neurons rapidly In the present study, we modified AFG with NCAD-Fc in vitro . In animal experiments, AFG was also proven to via biochemical conjugation to successfully obtain a novel facilitate axon regeneration and motor functional recovery in multifunctional scaffold AFG-NCAD-Fc (AFGN) to carry rodent and canine SCI models [23, 24]. However, AFG lacks exogenous NSCs for spinal cord regeneration [10, 38, 41]. specific biochemical cues to re-engineer the NSC desirable The AFGN hydrogel exerts synergistic multimodal bio- niche, which could coax the grafted NSCs’ retention and physical and biochemical cues, including linearly ordered neuronal differentiation. nanoscale topography, soft stiffness, and NCAD presented Biochemical cues, such as soluble neurotrophic factors biological activity, which worked together and eventually and cell adhesion molecules (CAMs), play critical roles rebuilt the favorable niche for the NSCs. Subsequently, in constructing stem cell niches, regulating stem cell fate, NSC adhesion and neuronal differentiation on the AFGN and accelerating axon regeneration and are widely applied hydrogel were evaluated in vitro. Furthermore, the AFGN in biomaterial functional modification as well as bioactive hydrogel carrying exogenous NSCs was implanted into a interface creation . In recent years, studies on cadher- rat 2 mm-long complete transected SCI model to bridge ins, an important family of CAMs, have made tremendous the stumps. Structural reconstruction, axon regeneration, 1 3 Advanced Fiber Materials neurogenesis, and functional recovery were investigated KBr pellets. To observe the microstructure of the AFG and based on morphological, histological, electrophysiologi- AFGN hydrogels, the hydrogels were fixed in cold 2.5% cal, and locomotor parameters. (v/v) glutaraldehyde for 3 h and dehydrated by an etha- nol series. After lyophilization for 24 h, the samples were characterized by scanning electron microscopy (SEM, Carl Zeiss, GEMINISEM 500, Germany). The binding rate of Experimental Section NCAD-Fc immobilized onto the AFGN was determined using a human N-cadherin ELISA Kit (MM-13240H2, Fabrication of AFGN Hydrogel MEIMIAN, China) by subtracting the concentration of uncombined NCAD-Fc from the initial concentration, as AFG was generated by electrospinning as previ - previously described . ously described [ 10]. In brief, 2% (w/v) fibrino - The rheological properties of the AFG and AFGN were gen (F8630, Sigma-Aldrich, USA) with 0.5% (w/v) examined using an 8 mm-diameter parallel plate at 37 ℃ poly(ethyleneoxide) (PEO, 4000 kDa, Sigma-Aldrich, (Physical MCR301 rheometer, Anton Paar GmbH, Graz, USA) in distilled water was electrospun and collected in Austria). The dynamic frequency sweep test (0.1–10 Hz at a liquid bath with 50 mM CaCl and 5 units/mL thrombin 1% strain) was used to record the storage (G′) and loss (G′′) (T4648, Sigma-Aldrich, USA) according to the follow- modulus. The stiffness of the AFG and AFGN hydrogels ing conditions: 23 G needle, a voltage of 4 kV, rotating was assessed by atomic force microscopy (AFM, Dimen- collector at a speed of 60 rpm. Subsequently, the AFG sion ICON, Bruker, Billerica, MA, USA). Force curves from fibers were assembled to form a 2 mm-thick bundle for 500 randomly chosen points per hydrogel were registered further uses. After removal of PEO from the AFG by using silicon SNL-D probes consisting of 20 μm-diameter washing with distilled water, the AFG was exposed to glass beads attached to the edge of a silicon nitride V-shaped ultraviolet light overnight for sterilization. The immobi- cantilever with a nominal spring constant of 0.06 N/m. The lization method was modified from protocols published Young’s moduli of the hydrogel surfaces were evaluated previously and performed under sterile conditions [38, from the force-indentation curves by fitting to the Hertz 41, 42]. The COOH groups of the AFG were activated model. The water contact angle of the AFG and AFGN was and stabilized in a solution of 0.4 mg/mL 1-ethyl-3- measured using an OCAH200 Optical contact angle tester (dimethylaminopropyl) carbodiimide hydrochloride (Dataphysics, German), as previously described . (EDC) (Sigma-Aldrich, USA)/0.6 mg/mL N-hydroxy- succinimide (NHS) (Sigma-Aldrich, USA) for 30 min at In Vitro NSC Isolation and Culture Experiment room temperature. The AFG was then rinsed three times with distilled water for 15 min. Afterward, the AFG was NSCs were isolated from fetal (embryonic Day 14) SD rats coated with 2 μg/mL protein A (PA) (21,181, Pierce, under sterile conditions, as previously described with slight USA) solution for 2 h at 37 ℃. Following three washes, modifications . In brief, the hippocampus of the embryo the AFG was finally soaked in 10 μg/mL recombinant was dissected on ice gently to strip the endocranium and human N-cadherin human Fc chimera protein (NCAD- blood vessels. After dissection, the hippocampal tissues were Fc, SOMAR, Japan) solution for 12 h at 37 ℃, form- transferred to a dish with ice-cold PBS and cut into small ing a novel functional hydrogel named AFG-NCAD-Fc pieces, digested in 0.05% EDTA/Trypsin (Gibco, USA) (AFGN). All procedures were performed under sterile at 37 ℃ for 10 min, and centrifuged at 200×g for 5 min. conditions. The cell suspension was cultured in neurobasal medium (Gibco, USA) containing 2% B27 (Gibco, USA), 1% peni- Characterizations of AFGN Hydrogel cillin–streptomycin (PS, Sigma-Aldrich, USA), 20 ng/mL epidermal growth factor (EGF, Solarbio, China), and 20 ng/ Proton nuclear magnetic resonance ( H NMR) was applied mL basic fibroblast growth factor (bFGF, Solarbio, China). to verify the conjugation of AFG and AFGN. The AFG and Afterward, NSCs were kept in an incubator containing 5% AFGN powders were dissolved in deuterium oxide (D O) CO at 37 °C, and half of the medium was changed every 2 2 2 at a concentration of 10 mg/mL. The H NMR spectra were days. Seven days later, the newly formed NSC spheres were recorded on a 600 M NMR spectrometer (JNM-ECA600, digested into single cells with Accutase (Invitrogen, USA). JEOL Ltd., Japan) at room temperature. For Fourier trans- Single NSCs were cultured using DMEM/F12 proliferative form infrared (FTIR) spectrometry, the freeze-dried AFG medium-containing 2% B27 (Gibco, USA), 1% N2 (Gibco, and AFGN hydrogels were ground into fine powders. The USA), 1% PS (Sigma-Aldrich, USA), 20 ng/mL EGF (Solar- −1 FTIR spectra were recorded from 4000 to 400 cm on bio, China), and 20 ng/mL bFGF (Solarbio, China). Cul- an FTIR spectrometer (X70, NETZSCH, Germany) using tured NSCs were collected for in vitro NSC adhesion and 1 3 Advanced Fiber Materials differentiation experiments. For in vivo transplantation, by an mRNA kit (Tiangen, China), and then reversed to the NSCs were incubated with DMEM/F12 proliferative cDNA using a FastQuant RT kit (Tiangen, China), for a medium-containing lentiviral particles (pLV-EGFP/Puro- quantitative real-time polymerase chain reaction (qRT-PCR). CMV, Cyagen Biosciences, China) and polybrene (5 μg/ The qRT-PCR was performed using SYBR Green super- mL) at a multiplicity of infection (MOI) of 50 for 16 h to mix (Bio-Rad, USA) and detected by the CFX96 real-time obtain GFP-labeled NSCs. In addition, some NSC spheres PCR detection system (Bio-Rad, USA). The relative gene at 7 days were randomly collected and stained with Nestin expression was normalized by the GAPDH and determined −△△Ct (1:100, sc-23927, Santa Cruz, USA) for the identification of by the 2 method . The primer sequences are listed NSCs, and images were taken with confocal laser scanning in Table S1. microscopy (LSM980 Airyscan2, Zeiss, Germany). The Immunoregulation Experiment in Vitro Seeding of NSCs onto the Hydrogels In Vitro The rat HAPI microglia cells (a generous gift from Profes- To identify NSC adhesion, single NSCs were resuspended sor Li-Na Zhang, Department of Critical Care Medicine, in DMEM/F12 culture medium-containing 1% PS (Sigma- Xiangya Hospital, Central South University, China) were Aldrich, USA) and 10% fetal bovine serum (FBS, Gibco, cultured in Dulbecco’s Modified Eagle’s Medium (high Australia) to promote NSC adhesion, and then seeded onto glucose, DMEM, Pricella) which contained 10% FBS and the AFG and AFGN hydrogels at a density of 1 × 10 cells 1% PS, in an incubator containing 5% C O at 37 °C. The per scaffold and cultured in an incubator containing 5% CO HAPI cells were seeded on the TCP control (TCP group) and 5 2 at 37 °C. After culturing for 1 day, 3 days, and 7 days, the AFGN hydrogel (AFGN group) at a density of 1 × 10 /cm . samples were gently washed with PBS and then fixed in 4% Meanwhile, the HAPI and NSC cells (5 × 10 cells/insert) paraformaldehyde (PFA) for 1 h at 4 °C. Subsequently, the were separately seeded into a 24‐well transwell system NSCs retained on the hydrogels were permeabilized with (Corning, 0.4 μm pores, USA) (NSC group). Followed by 0.1% Triton X-100 for 20 min and blocked with 10% nor- adhesion for 12 h, the HAPI cells were stimulated by adding mal goat serum (Solarbio, China) for 1 h. Then, the samples 1 μg /mL lipopolysaccharide (LPS, Invivogen, France) and were incubated with rhodamine phalloidin (RP, Solarbio, 20 ng/mL interferon-γ (IFN-γ, adcam, UK) in a DMEM-F12 China) and DAPI (sc-74421, Santa Cruz, USA) for 40 min. medium for 24 h to polarize. Subsequently, the total RNA Images were captured by confocal laser scanning micros- was extracted from the HAPI cells of all groups, respec- copy (LSM980 Airyscan2, Zeiss, Germany). Meanwhile, tively, and analyzed by the qRT-PCR assay. The primer the NCAD-Fc release during the cell culture process was sequences are listed in Table S1. assessed. The culture medium of NSCs on the AFGN was collected and refreshed every day. An ELISA kit was used to Animal Procedures quantify the released concentration of NCAD-Fc. To identify NSC differentiation, single NSCs were resus - Animal experiments were performed in accordance with the pended in the DMEM/F12 culture medium-containing 1% Guide for the Care and Use of Laboratory Animals formu- PS and 10% FBS to promote NSC differentiation and then lated by the National Institutes of Health (NIH) and were seeded onto the AFG and AFGN hydrogels at a density of approved by the Institutional Animal Care and Use Com- 1 × 10 cells per scaffold. Moreover, the NSCs seeded on the mittee of Tsinghua University (Beijing, China). Healthy TCP in the culture medium with or without 50 ng/mL nerve female SD rats (8 weeks old, 200–230 g, n = 104) were used growth factor (Solarbio, China) as for positive control (PC) in this study. Surgeries were performed under anesthesia or negative control (NC) group, respectively. After 7 days with an intraperitoneal injection of 1% w/v pentobarbital of culture at 37 °C, the samples were washed with PBS, sodium solution (30 mg/kg) to establish the rat SCI model. fixed in 4% PFA, permeabilized by 0.1% Triton X-100, and Briefly, a dorsal laminectomy was performed to remove the blocked with 10% normal goat serum sequentially. Then, the T8–T10 vertebrae. Under a microscope, a 2 mm-long block samples were immunostained with Tuj-1 (1:500, ab18207, of the T9 spinal cord was completely transected and removed Abcam, UK) and GFAP (1:500, ab4648, Abcam, UK) pri- using microscissors. The ventral dura mater was preserved, mary antibodies for 16 h at 4 °C, followed by incubation and gel foam was used for hemostasis. All animals were with corresponding secondary antibodies and DAPI at room randomly divided into four groups according to different temperature for 40 min. Images were taken with a confo- treatments. AFGN hydrogel (AFGN group), 10 μL of cell cal laser scanning microscope (LSM980 Airyscan2, Zeiss, suspension containing 1 × 10 GFP-labeled NSCs (NSC Germany). group), 10 μL of saline (control group), or AFGN hydrogel Furthermore, after 7 days of culture, the total RNA of with 1 × 10 GFP-labeled NSCs (AFGN-NSC group) was NSCs from the NC, PC, AFG, and AFGN group was isolated immediately implanted into the lesion site to fill the gap. 1 3 Advanced Fiber Materials Finally, the surgical incisions were closed with sutures. After Functional Behavior Analysis the operation, all animals received meloxicam (4 mg/kg) for 3 days and penicillin for 7 days. Manual bladder extrusion During the 12 week repair period, locomotor function was was applied three times a day until automatic micturition evaluated weekly by two independent observers blinded to recovered. the conditions using the Basso–Beattie–Bresnahan (BBB) rating scale . The rats (n = 8 for each group at each time Histological Evaluation point) were placed into an open field (100 cm diameter) and observed for 5 min. The hindlimb movements of each rat At 1 week, 4 weeks, 8 weeks, and 12 weeks postsurgery, rats were scored and recorded. Moreover, the gait was recorded were sacrificed and consecutively perfused with saline and at 12 weeks postsurgery using Catwalk XT 10.6 System 4% w/v PFA after pentobarbital anesthesia, and the spinal (Noldus, Wageningen, The Netherlands) . CatWalk cord tissue segments containing the lesion epicenter were 10.6 software labeled paw prints automatically for further collected. Moreover, bladder, heart, liver, spleen, lung, and analysis. kidney tissues were also harvested. Tissue samples were fixed in 4% w/v PFA at 4 °C overnight and successively Electrophysiological Evaluation placed into sucrose solution (30% sucrose in 0.1 M PBS) at 4 °C for 72 h. Tissue samples were separately embedded Motor-evoked potentials (MEPs) were carried out using in OCT compound and sliced into 10 μm-thick histologic Electromyograph and Evoked Potential Equipment (33A07, sections using a cryostat microtome (CM 1950, Leica, Ger- Dantec Dynamics, Denmark). All rats received the same many). These tissue sections were used for hematoxylin and anesthesia procedure during surgery and electrophysiologi- eosin (H&E) staining, Masson staining, and immunofluo- cal evaluation. The transcutaneous bipolar needle electrodes rescence staining. Images of H&E or Masson staining were were positioned on the skull surface to induce MEPs. The taken with a Pannoramic SCAN scanner (3DHIESTECH, recording needle electrodes were placed into the tibialis Hungary) and processed using CaseCenter 2.9SP1 software anterior muscle, and then, the grounding electrode was (3DHIESTECH, Hungary). For immunofluorescence stain- placed into the back subcutaneously. The peak amplitude ing, the sections were rinsed with PBS and then immersed and latency of the MEPs were measured and compared in PBS containing 10% w/v normal goat serum and 0.3% among the groups. w/v Triton X-100 for 2 h at room temperature. Then, the sections were incubated with primary antibodies (Table S2) Statistical Analysis at 4 °C overnight, washed with PBS, and incubated with the corresponding secondary antibodies (Table S1) for 1 h in Statistical analysis was carried out in SPSS Statistics for the dark. DAPI (Abcam, UK) mounting medium was used Windows (v.23.0; IBM Corp., USA). All data are presented to stain cell nuclei. Images of immunofluorescence stain- as the mean ± standard deviation (SD) unless otherwise indi- ing were collected on a Zeiss Axio Scan Z1 scanner (Carl cated. One-way analysis of variance (ANOVA) followed by Zeiss, Germany) and processed using Zen 2.6 (Blue edition) LSD (equal variances) or Dunnett’s T3 post hoc test (une- software (Carl Zeiss, Germany) under the same parameters. qual variances) was performed to determine significant dif- ImageJ 1.51 k (Wayne Rasband, NIH, USA) was used for ferences for multiple comparisons. Two-way repeated-meas- quantitative analysis of the representative immunofluores- ures ANOVA was used to compare significant differences cence and H&E images. between each group and time point in the BBB experiment. At 12 weeks postoperatively, the lesion epicenters in the All tests were two-sided, and a P value < 0.05 indicated a other 12 rats (3 rats for each group) were retrieved and sliced statistically significant difference. into 60 nm-thick ultrathin axial sections or 500 nm-thick semithin axial sections using a microtome (EM UC6, Leica, Germany) for further detection of myelination, axon regen- Results and Discussion eration, and synapse ultrastructure [24, 47]. The semithin sections were stained with toluidine blue/borax solution and Fabrication of AFGN Hydrogel scanned using a Pannoramic SCAN scanner (3DHIESTECH, Hungary). The ultrathin sections were doubly stained with AFG was generated by electrospinning as described in our lead citrate and uranyl acetate, and images were captured previous studies . Afterward, NCAD-Fc was immobi- by a transmission electron microscope (TEM, Tecnai Spirit, lized onto AFG via biochemical conjugation, forming the FEI, Czech Republic). ImageJ 1.51 k (Wayne Rasband, NIH, functionalized AFGN hydrogel (Fig. 1a and S1a). Com- USA) was used for quantitative analysis of the representative pared with chemical crosslinking, biochemical conjugation toluidine blue staining and TEM images. 1 3 Advanced Fiber Materials with PA is more advantageous in preserving the function could bind to each other through homophilic interaction to of NCAD-Fc . The successful conjugation of NCAD- construct the structure of focal adhesion (FA) and adher- Fc to the AFG was examined by H NMR (Fig. 1b) and ens junction (AJ), which are necessary conditions to medi- FTIR spectrometry (Fig. 1c). The H NMR spectra of AFGN ate cell–ECM interaction and cell–cell adhesion [29, 33]. showed extra peaks at 2.5 ppm corresponding to the intro- Meanwhile, the intracellular regions of NCAD could interact duction of a characteristic methylene group by NCAD-Fc, with p120 catenin and indirectly bind to the actin filaments which was absent in AFG. In addition, the FTIR spectra (F-actin), which enables cells to sense and feedback to the −1 showed characteristic peaks at 1639 cm in the AFGN, biophysical cues of the substrate and modulates cellular reflecting the preserved β-sheet structure due to biochemical matrix mechanosensing through the FAK/AKT/YAP path- conjugation. SEM images showed nanoscale aligned fibrous way [25, 33]. Therefore, we conducted qRT-PCR to evalu- structures in both AFG and AFGN hydrogels (Fig. 1d and ate the relevant gene expression in NSCs after culturing S1b), exerting biophysical cues by mimicking the linearly 7 days on different hydrogels. As shown in Fig. S3, the gene ordered ECM in the spinal cord. The rheological measure- expression of the adhesion specific markers (YAP, p120, ment results indicated that the AFG and AFGN hydrogels AKT, and Actin) exhibited greater up-regulation in AFGN remained steady under constant deformation and exhibited group in comparison to AFG group. Our findings are in similar viscoelasticity properties (Fig. 1e, f), which were agreement with the previous studies, indicating that AFGN close to the viscoelasticity of soft tissue (tanδ∼10–20%) could significantly promote cell adhesion, modulate cellular . The Young’s moduli of the AFG and AFGN hydro- matrix mechanosensing, and adjust the actin cytoskeleton in gels were 1.22 ± 0.17 kPa and 1.18 ± 0.06 kPa (Fig. 1g), response to the aligned nanoscale topographies of the fiber respectively, which were similar to the moduli of the ECM in matrix and promote the cell directional elongation. native nerve tissue (∼1–4 kPa) [50–52]. The ELISA results Furthermore, we examined the differentiation potential demonstrated that the binding rate of NCAD-Fc immobi- of NSCs in the NC, PC, AFG, and AFGN groups using lized onto the AFGN hydrogels was 28.1 ± 4.6% (Fig. S1c). Tuj-1 (β-tubulin III, early neuron marker) and glial fibril- The water contact angles of the AFG and AFGN were lary acidic protein (GFAP, astrocyte marker) antibodies 65.90 ± 4.70° and 50.87 ± 8.08°, respectively (Fig. S1d). after 7 days of culture. As shown in Fig. S4a, compared These results demonstrated that the NCAD-Fc was success- with the NC group, cells of PC group exhibited signifi- fully immobilized onto the AFGN hydrogels and enhanced cantly increased positively stained for Tuj-1 with the the hydrophilicity of the hydrogel. induction of nerve growth factor. However, cells of both NC and PC groups were oriented in random directions. In Vitro Modulation of NSC Adhesion In contrast, cells on the AFG showed positive staining and Differentiation by the AFGN Hydrogel for both Tuj-1 and GFAP with a certain degree of elon- gation along the direction of the fibers, whereas cells NSCs were isolated from the hippocampus of embryonic on the AFGN exhibited increased, obviously elongated SD rats . After culturing for 7 days, the newly formed and oriented Tuj-1-positive staining with relatively weak NSCs neurospheres (Fig. S2a) were identified by Nes- GFAP expression. Meanwhile, qRT-PCR were conducted tin (NSC marker). As expected, the cells in neurospheres to evaluate the relevant gene expression in NSCs after cul- were almost totally Nestin-positive (Fig. S2b), confirming turing on the different substrates (Fig. S4b). It was shown their NSC nature. To investigate NSC adhesion on different that in the AFGN group, the gene expressions of the neu- hydrogels, NSC neurospheres were digested into single cells ronal lineage markers (Tuj-1, NSE, and NeuN) were sig- and seeded onto the AFG and AFGN hydrogels. Interest- nificantly increased when compared with other groups, ingly, we observed obvious differences in cell morphology but the MAP-2 (mature neuron marker) gene expression on the AFG and AFGN. Starting from day 1 after cell seed- showed no statistical difference between the AFG and ing, the cytoskeletons of cells cultured on the AFGNs began AFGN group. Moreover, compared with the NC and AFG to elongate along fibrin nanofibers, while those on the AFGs group, the GFAP gene expression decreased obviously in still showed inconspicuous prior orientation. Over time, we the AFGN group. These results indicated that AFGN could observed that AFGN remarkably enriched more cells than promote the neuronal differentiation of NSCs in vitro AFG, and cells on the AFGN presented increasingly directed through the enhanced cell–ECM interaction and cell–cell elongation on day 3. After 7 days of cell culture, abundant adhesion as well as adhesion-mediated mechanical feed- cells overlying the surface of AFGN exhibited impressive back . time-evolving elongated morphology and well-developed In addition, the release of NCAD-Fc from AFGN during cytoskeleton actin filaments that highly coaligned with the the cell culture was also investigated by ELISA. As shown long axial direction of AFGN nanofibers (Fig. 1h). Based on in Fig. S5, comparing with the initial binding concentra- previous research, the extracellular domains of N-cadherin tions of NCAD-Fc detected, more than 97% NCAD-Fc was 1 3 Advanced Fiber Materials Fig. 1 The fabrication and characterization of the AFGN hydrogel. a Young’s moduli of AFG and AFGN hydrogels measured by atomic Schematic diagram of the fabrication of AFGN hydrogel, b H NMR force microscopy (AFM). *P < 0.05; n.s. no significant; n = 3, and h spectra of AFG and AFGN, c representative FTIR spectra of AFG representative cytoskeleton F-actin (stained with rhodamine phalloi- and AFGN hydrogels, d SEM images of AFG and AFGN hydrogels, din, red) staining images of NSCs on AFG and AFGN cultured for e rheological measurements of storage (G′) and loss (G′′) modulus 1 day, 3 days, and 7 days, respectively. The nuclei were stained with of AFG and AFGN, f tanδ (G′′/G′) at 1 Hz of AFG and AFGN, g DAPI (blue). Scale bar represents 100 μm remained on the AFGN after culturing for 7 days, indicating In Vivo Implantation and Motor Functional Recovery that the NCAD-Fc can be stably immobilized on the AFGN Evaluations hydrogels. The effects of the AFGN hydrogel on spinal cord regenera- tion and NSC desirable niche reconstruction were evaluated through implantation in a rat 2 mm T9 complete transection 1 3 Advanced Fiber Materials SCI model (Fig. S6). After SCI, the rats were kept for no obvious increase during the 12 weeks. In particular, at 12 weeks (Fig. 2a) and divided into four groups as follows: 12 weeks postsurgery, rats in the AFGN-NSC group could rats that received AFGN hydrogel implantation (AFGN make occasional weight-supported plantar steps (Fig. 2d, e) group), rats that received GFP-labeled NSC implantation and forelimb–hindlimb coordination (Fig. 2e), whereas in (NSC group), those that received GFP-labeled NSCs carried the NSC and control groups, the rats displayed persistent by AFGN hydrogel (AFGN-NSC group), and the control dragging of the hindlimbs without weight support. group (merely received saline) (Fig. 2b). To evaluate the Motor function recovery was further confirmed by per - locomotor function recovery of hindlimbs, the BBB rating forming electrophysiological analyses. The cortical MEPs test was performed weekly after surgery, and the paw prints exhibited approximately 2.1 mV amplitude and 3.0 ms of the rats at 12 weeks postsurgery were recorded using Cat- latency before SCI and disappeared completely after sur- Walk XT 10.6 System. From 2 weeks postsurgery, the mean gery, indicating that the rat SCI model was successfully BBB score of the AFGN-NSC and AFGN groups exhibited a established. Although none of the four groups recovered significant improvement, especially the AFGN-NSC group, to normal levels at 12 weeks after surgery, a significantly to almost 9 at 12 weeks postsurgery (Fig. 2c). However, shorter latency and higher amplitude were detected in the the BBB scores of the NSC and control groups displayed AFGN-NSC group than in the other groups (Fig. 2f, g), Fig. 2 Functional and electrophysiological recovery evaluations normal rats, rats immediately after surgery and of each group, and g in vivo. a Timeline of the experiments in vivo, b schematic of differ - quantitative analysis of MEPs latency and amplitude. Data are pre- ent graft-based grouping of the experiments in vivo, c the BBB scores sented as the mean ± standard deviation (SD). *P < 0.05, **P < 0.01, of rats in each group, *P < 0.05, **P < 0.01, ***P < 0.001; n = 8, d, ***P < 0.001; n.s. no significant; n = 3; h specimens of the spinal e typical records of rat walking gaits (d) and the Catwalk footprint cords of all experimental groups 12 weeks postsurgery; the black rec- depiction (e) 12 weeks postsurgery, f representative MEPs traces of tangles indicate the lesion sites 1 3 Advanced Fiber Materials which provided electrophysiological evidence for motor for AFGN-NSC vs. control and NSC, respectively; n.s. for function recovery attributed to AFGN-NSC implantation. AFGN-NSC vs. AFGN) (Fig. 3a, b). However, there was no At 12 weeks postsurgery, the physical tissue repair of statistical difference between the AFGN and AFGN-NSC the spinal cord and bladder was also evaluated. As shown group in the inhibitory effect on inflammation. The results in Fig. 2h, the lesion epicenters of the spinal cord tissue in of qRT-PCR in vitro showed a similar trend as immunostain- the control and NSC groups remained empty. In the AFGN ing (Fig. S9). The expression of inflammatory gene Iba1 in group, the gaps were partially filled by the newly regenerated the TCP control group was significantly higher than other tissues with rough and irregular surfaces, whereas those in groups. The expression of proinflammatory genes TNF-α the AFGN-NSC group almost occupied the majority of the and CD86 in the AFGN group decreased obviously when lesion sites and presented good integration with the host compared with other groups, but the expression of TNF-α spinal cord. showed no statistical difference between the TCP control and Neurogenic bladder is one of the most fatal complications NSC group. Moreover, compared with the other two groups, of SCI . Therefore, the protection and repair of bladder the expression of anti-inflammatory gene Arg-1 showed a tissue is a therapeutic challenge during SCI recovery. We significantly upward trend in the AFGN group. These results found that the enhanced regeneration of the spinal cord in could be explained by the fact that the AFGN hydrogel could the AFGN-NSC group displayed a synergistic benefit for exert immunomodulation function to regulate the infiltration the protection and recovery of bladder tissue (Fig. S7). In of macrophages and shift activated macrophages into the the control group, the bladder wall exhibited severe thick- M2 subtype even without NSCs, possibly due to its suitable ening and fibrosis changes, along with muscle atrophy and physicochemical properties and the nanostructure–function edema of the mucous membrane. However, the pathological relationship, in agreement with the previous studies [56–58]. damage of the bladder tissue was successfully restricted in It is notable that the NSCs also had a certain degree of alle- the AFGN-NSC group. These results, combined with BBB, viating inflammation both in vivo and in vitro, which might CatWalk, and electrophysiological evaluations, demon- be due to the complex crosstalk between the activated mac- strated that the grafted NSCs carried by the AFGN hydrogel rophages and the various cytokines secreted by NSCs . improved tissue repair and functional recovery after SCI. The acute inflammatory storm induced by activated M1 In addition, the H&E staining images of the hearts, livers, macrophages after SCI can cause syringomyelia and trigger spleens, kidneys, and lungs exhibited no obvious cellular reactive scar-forming astrocytes to deposit axon-inhibitory toxicity and pathological differences in any of the groups chondroitin sulfate proteoglycan (CSPG) [24, 60], leading (Fig. S8), which demonstrated reliable biocompatibility of to the formation of glial scars and chronic failure of axon the implanted NSCs and AFGN hydrogel . regeneration. As shown in H&E staining images (Fig. 3c), dense glial scars and obvious cavities were found in the con- Delivery of NSCs via the AFGN Hydrogel Regulates trol group. In the NSC group, a few regeneration tissues the Local Immune Environment and Reduces randomly grew into the SCI area. Without the mechanical Inflammation‑Triggered Secondary Injury support of the scaffold, grafted NSCs have previously been shown to tend to spread into the surfaces of surrounding tis- The spinal cord tissue sections were further examined to sue as a result of the incongruousness in mechanical strength confirm the function of the AFGN hydrogel-NSC system in . Moreover, without the shelter created by the scaffold, promoting SCI repair and functional recovery. The neuroin- the grafted NSCs were directly exposed to the hostile niche, flammation induced by recruited macrophages is well docu- in which the infiltrated macrophages could secrete cytotoxic mented to reach a peak at the lesion site 7 days after acute factors to attack foreign bodies [62–64]. These processes SCI, resulting in a cascade of secondary injury processes may cause the loss and apoptosis of grafted NSCs, resulting . Activated macrophages can bind to specific factors and in the formation of glial scars and cavities that are similar polarize into the M1 subtype (exert proinflammatory func- to those in the control group . In the AFGN and AFGN- tion) and M2 subtype (exert anti-ina fl mmatory function). To NSC groups, many newborn tissues permeated into the examine the effect of AFGN-NSC transplantation on inflam- injured site with a scaffold-oriented aligned direction. On matory regulation, immunostaining for CD68 (a marker of the other hand, the mean volume of cavities was significantly activated macrophages) and CD206 (a marker of M2 mac- reduced in the AFGN-NSC group compared to the other rophages) was performed 7 days after injury. We found that groups (P < 0.001 for AFGN-NSC vs. control and NSC, abundant activated macrophages accumulated at the lesion respectively; P < 0.01 for AFGN-NSC vs. AFGN) (Fig. 3c, site (Fig. 3a), while treatment with AFGN or AFGN-NSC d). Moreover, we performed immunostaining for CS-56 (a noticeably reduced the number of activated macrophages marker of CSPG) to assess CSPG deposition 12 weeks after and presented large quantities of M2 macrophages in the SCI. In the control group, obvious CSPG deposition with injured area compared with the other groups (P < 0.001 a wall-like structure filled the epicenter of the lesion site 1 3 Advanced Fiber Materials Fig. 3 Modulation of local inflammatory response and inflammation- lesion site and the host spinal cord; the black triangles indicate cav- triggered secondary injury. a Representative images of CD68 (green)/ ity formation; the black arrows indicate glial scar, d Quantification of CD206 (red)/DAPI (blue) immunostaining at the lesion site and the the cavity of area (cavity area/total area). Data are presented as the amplified images of each group at 1 week postgrafting. b Quantifica- mean ± (SD); **P < 0.01, ***P < 0.001; n.s. no significant; n = 5, tion of the density of CD68-positive and CD68/CD206 dual-positive e Representative immunostaining images of CS-56 from different cells. ***P < 0.001; n = 5, c Representative images of H&E staining treatment groups 12 weeks postsurgery, e2, e4, e6, e8 The enlarged (nuclei: blue, cytoplasm: red) for longitudinal sections of each group, images of the boxed regions in e1, e3, e5, e7, respectively; the dotted 4 weeks postgrafting, c2, c4, c6, c8 Enlarged images from c1, c3, c5, lines indicate the boundaries between the lesion site and the host tis- c7, respectively; the dotted lines indicate the boundaries between the sue and surrounded the lesion boundary. Conversely, only spo- glial scars in scenarios of secondary injury processes, paving radic CSPG deposition was found in the AFGN-NSC group the way for SCI repair. (Fig. 3e). Collectively, our results suggest that the AFGN hydrogel in combination with NSCs regulates the local neu- The AFGN Exerts NSC‑Protective Eec ff ts roinflammation niche by shifting activated macrophages into and Directed Neuronal Differentiation of Grafted the M2 subtype after acute SCI. The modified early stage NSCs immune microenvironment further alleviated syringomy- elia and reduced the deposition of CSPG at the lesion site, The inhospitable niche at the lesion site after SCI and the achieving a long-term effect on mitigating the formation of lack of adhesive cues cause low-grafted NSC retention and neuronal differentiation but facilitate glial lineage 1 3 Advanced Fiber Materials differentiation, which constitutes the main barrier to NSC- (P < 0.001) (Fig. 4a2, b). Meanwhile, the AFGN-NSC based SCI therapies [4–6]. To investigate whether the AFGN group exhibited a significantly higher fraction of GFP/Tuj-1 could induce NSC retention and neuronal differentiation double-positive area as well as a lower fraction of GFP/ in vivo in the middle stage of SCI, immunostaining of Tuj- GFAP double-positive area than the NSC group (P < 0.001) 1, GFAP, and GFP were used to label newborn neurons, (Fig. 4a1, a2, c), indicating that the AFGN hydrogel could astrocytes, and grafted NSCs, respectively (Fig. 4a). Four promote the neuronal differentiation of grafted NSCs and weeks postgrafting, abundant GFP-positive NSCs were inhibit glial differentiation. Moreover, the AFGN-NSC detected at the lesion site in the AFGN-NSC group, effec- group remarkably boosted Tuj-1-labeled neuron regenera- tively preventing the loss of grafted NSCs (Fig. 4a1). Con- tion manifesting tubules in the central lesion compared with versely, the grafted NSC density (ratio of GFP-positive area other groups (P < 0.01) (Fig. 4a, d). These results could be to total area) in the NSC group was significantly decreased explained by the fact that the NCAD could enhance the Fig. 4 Retention and neuronal differentiation of grafted NSCs and tively, b quantification of GFP-positive area from the enlarged view, c neural regeneration. a Representative images of Tuj-1 (red), GFAP quantitative analysis of the GFP/Tuj-1 and GFP/GFAP dual-positive (white), and DAPI (blue) immunostaining for longitudinal sections of area from the enlarged view, and d quantification of Tuj-1-positive each group at 4 weeks postgrafting. The implanted NSCs are iden- area from the enlarged view. Data are presented as the mean ± (SD); tified by GFP fluorescence signals (green) in a3–a4. a11–a13, a21– *P < 0.05, **P < 0.01, ***P < 0.001; n = 5 a23, a31–a33, a41–a43 Enlarged images from a1, a2, a3, a4, respec- 1 3 Advanced Fiber Materials mechanotransduction of the cell–biomaterial interface higher in the AFGN-NSC group than in the other three and mediate NSCs to better perceive the biophysical cues groups (P < 0.001), approaching those of normal spinal cord generated by the AFGN hydrogel, leading to an enhance- tissue but with a significant difference (P < 0.001) (Fig. 5c, ment retention of grafted NSCs in the lesion site through d). Furthermore, TEM images were used to examine the the cell–biomaterial and cell–cell adhesive contacts [27, 28, ultrastructural details of the myelin sheath, including the 30, 31]. In addition, previous studies have shown that NCAD myelin sheath thickness and myelinated axon area. In the can also exert biochemical cues to reduce the level of cyto- AFGN-NSC group, the myelin sheath was significantly plasmic β-catenin, which promotes the neural differentia- thicker than those in the other three groups (P < 0.01), and tion of grafted NSCs through the Wnt/β-catenin and AKT the area-based G-ratio (axon area/whole myelinated axon signaling pathways [34, 35]. Taken together, the AFGN area) was significantly lower than those in the other three hydrogel could significantly promote adhesion and neural groups (P < 0.05); in both cases, the values in the AFGN- differentiation of grafted NSCs in the SCI area, demonstrat - NSC group were closest to those in normal spinal cord tis- ing an efficient desirable niche reconstruction capacity of sue but with significant differences (Fig. 5e, f). Moreover, the AFGN hydrogel. the structure of myelin sheath-wrapped axons evaluated by immunofluorescence for neurofilament 200 (NF, a marker NSC Delivery via the AFGN Hydrogel Facilitates of mature neurons)/myelin basic protein (MBP, a marker of Angiogenesis and Axon Remyelination myelin sheath) was also more obvious in the AFGN-NSC group compared with other groups (Fig. 5g and S10), which The process of angiogenesis after SCI is essential for axonal is consistent with the previous results. Taken together, these regeneration and long-term survival, because it is crucial for results indicate remarkable axon regeneration and mature the supply of nutrients and oxygen . To better evaluate myelin regeneration mediated by the synergistic effect of the the vascularization effects of the AFGN hydrogel loaded AFGN hydrogel and grafted NSCs, verifying the previously with NSCs, we performed immunostaining for Tuj-1 and reported mechanisms of the initial axon–oligodendrocyte RECA-1 (a marker of vascular endothelial cells) 8 weeks contact myelination process and NSC systematic differen- after SCI (Fig. 5a). Only a few sporadic Tuj-1- and RECA- tiation induced by NCAD [26, 69, 70]. 1-labeled cells could be seen at the lesion site in the control group and the NSC group (Fig. 5a1, a2). In contrast, the Delivery of NSCs via the AFGN Hydrogel Promotes AFGN group exhibited enhanced neurogenesis accompanied Neural Relay Formation and Functional Neurons’ by balanced vascularization (Fig. 5a3), which may be due Regeneration to NCAD-induced neurite outgrowth  and the capac- ity of fibrin to promote endothelial cells to form functional The transplanted exogenous NSCs could differentiate into vasculature . Notably, the AFGN-NSC group promoted neurons and form synaptic connections with host neurons a significantly larger number of Tuj-1-labeled regrowth at the lesion site, which is considered to be crucial for neu- axons in directional arrangement synergizing with RECA- ral relay reconstruction and functional recovery after SCI 1-labeled lumen-like matured vessel structures, which were . To investigate the long-term fate of grafted NSCs, more obvious than those in the AFGN group (Fig. 5a3, a4). immunostaining for GFP, NF, and SYN was used to vis- These findings suggest that the NSCs in the AFGN hydrogel ualize grafted NSCs, neurofilaments (a marker of mature might exert paracrine ee ff cts and synergize with the scao ff ld, neurons), and synapses, respectively (Fig. 6a), at 12 weeks providing a hospitable microenvironment for long-term axon postsurgery. The immunostaining results showed that the regeneration and vascularization after SCI . AFGN-NSC group remarkably boost directional neurofila- The myelin sheath is required for normal axon develop- ment regeneration at the lesion site (Fig. 6a4), which was ment and maintenance and plays a critical role in supplying of great significant superiority compared with other groups axon-needed nutrition and accelerating axonal signal con- (P < 0.001) (Fig. 6a, b). In the AFGN-NSC group, many duction . The regenerated tissues in all groups were har- GFP-positive NSCs were evenly dispersed at the lesion vested and sliced transversely at the epicenter of the lesion site and migrated into the adjacent host spinal cord tissue. site for the evaluation of axon regeneration and remyelina- Moreover, high-density GFP and NF double-positive signals tion 12 weeks after SCI. The toluidine blue staining images were also observed both in the injured area and adjacent host revealed many regrowing axons wrapped in myelin sheaths tissue (Fig. 6a4). However, in the NSC group, such positive in the AFGN-NSC group. In comparison, such structures signals were greatly attenuated in the absence of the AFGN were much less prevalent in the AFGN group and sporadi- hydrogel (P < 0.001) (Fig. 6a3, c). These results demonstrate cally appeared in the NSC and control groups (Fig. 5b). that the AFGN hydrogel could provide a favorable niche for Quantitative analysis suggested that both the density of the implanted NSCs and continuously exert differentiation nerve fibers and myelinated nerve fibers were significantly 1 3 Advanced Fiber Materials Fig. 5 Revascularization and axonal remyelination. a Representative ing images, d quantitative analysis of the density of myelinated nerve images of Tuj-1 (red)/RECA-1 (green)/DAPI (blue) immunostain- fibers from toluidine blue staining images, e quantitative analysis ing at the lesion site of each group at 12 weeks postgrafting, b rep- of the myelin sheath from TEM images, and f quantitative analysis resentative toluidine blue staining and TEM images of regenerated of the area-based G-ratio from TEM images. *P < 0.05, **P < 0.01, myelin in uninjured sites and the lesion core of the control, NSC, ***P < 0.001; n.s. no significant; n = 3; g representative images of NF AFGN, and AFGN-NSC groups at 12 weeks postgrafting, c quantita- (green)/MBP (red) immunostaining at the lesion core of the AFGN- tive analysis of the density of nerve fibers from toluidine blue stain- NSC group at 12 weeks postgrafting, g2–g4 Enlarged images from g1 1 3 Advanced Fiber Materials Fig. 6 Neural relay formation. a Representative images of NF (red), NF/SYN copositive area from the enlarged view, d Quantitative anal- SYN (white), and DAPI (blue) immunostaining for longitudinal sec- ysis of the NF/SYN dual-positive area from the enlarged view. Data tions of each group at 12 weeks postgrafting. The implanted NSCs are presented as the mean ± (SD); **P < 0.01, ***P < 0.001; n.s. no are identified by GFP fluorescence signals (green) in a3–a4; a11– significant difference; n = 5, e TEM images of the synapses at the a13, a21–a23, a31–a33, a41–a43 Enlarged images from a1, a2, a3, lesion core in the AFGN-NSC group; the yellow arrows indicate the a4, respectively, b Quantitative analysis of the NF-positive area from ultrastructure of synapses the enlarged view, c Quantitative analysis of the GFP/NF and GFP/ information for the regeneration of nerve fibers derived from copositive nerve fibers (Fig. S11a) and GFP/NF/5-hydroxy - the grafted NSCs. tryptamine (5-HT) copositive nerve fibers (Fig. S11b). This Moreover, in the AFGN-NSC group, many NF-positive finding indicated partial NSC-derived neuronal differen- neurofilaments migrated from the lesion boundary into tiation into motor functional neurons and the restoration of the injured area and were in dense contact with GFP/NF- nerve circuits [47, 71, 72]. These results demonstrated that copositive neurofilaments (Fig. 6a4). Copositive immuno- the synergistic action of AFGN hydrogel loaded with NSCs fluorescence signals of GFP/NF/SYN and NF/SYN indicated induced neuron regeneration, neural relay formation, and abundant newly formed synapses between the grafted NSC- nerve circuit reconstruction when transplanted after SCI. derived neurons and newly regenerated host axons in the Additionally, the effects of the separate AFGN hydrogel AFGN-NSC group, which revealed a significant difference and exogenous NSC implantation were also examined and compared with other groups (P < 0.001) (Fig. 6a, d). The showed relatively limited improvement in neural repair. The existence of reconstructed synapses was also confirmed by AFGN hydrogel could exert biophysical and biochemical TEM (Fig. 6e). In addition, the AFGN-NSC group exhib- cues to guarantee that implanted NSCs are evenly retained ited significant functional nerve fibers regeneration at the in a favorable niche and differentiate into neurons for a long lesion site, including GFP/NF/tyrosine hydroxylase (TH) time, provide directional guidance to the implanted NSCs 1 3 Advanced Fiber Materials microglia cells preparation and transportation. We also thank Yue-teng and regenerated neurons, and enhance the efficiency of the Wei from Bruker Corp. for the help of atomic force microscope opera- functional integration between exogenous NSC-derived tion. Schemes and Table of Contents (TOC) are created with BioRen- interneurons and host neurons, paving the way for the for- der.com. This work was financially supported by the National Natural mation of correct synaptic connections and nerve circuit Science Foundation of China (Grant Nos. 32271414 and 82201521), the Tsinghua Precision Medicine Foundation (Grant No. 2022TS001), reconstruction [73, 74]. Moreover, NCAD could mediate and the National Key Research and Development Program of China synaptic adhesion by regulating the level of α-amino-3- (Grant No. 2020YFC1107600). hydroxy-5-methyl-4-isoxazole propionic acid in the postsyn- aptic region, which could further enhance synaptic plasticity Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. and stability , contributing to functional recovery. Overall, compared to the previous scaffold-based NSC Declarations transplantation strategy, we orchestrated the advantages of the nanostructure presented by fibrin-based scaffold and the Conflict of Interest The authors declare that there are no conflicts of biochemical effects of NCAD-Fc to determine cell fate of interest. delivered NSCs in a favorable niche, proposing an effective Open Access This article is licensed under a Creative Commons Attri- combinatorial therapy in SCI repair. bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes Conclusions were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in In summary, we designed and fabricated an NCAD-func- the article's Creative Commons licence and your intended use is not tionalized aligned fibrin nanofiber hydrogel that provided permitted by statutory regulation or exceeds the permitted use, you will both biophysical and biochemical cues as a superior niche need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . for the delivery and regulation of exogenous NSCs. The AFGN hydrogel could significantly promote the adhesion and neuronal differentiation of NSCs via enhanced cell–cell communication and cell–biomaterial interactions. More References importantly, the AFGN exhibited an outstanding capacity for reconstructing favorable niche to effectively promote the 1. Jazayeri SB, Beygi S, Shokraneh F, Hagen EM, Rahimi-Movaghar long-term preservation, integration, and cell fate regulation V. 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J Neurosci. 2010;30:16343. enzyme-mimics for catalytic therapeutics and diagnostics. Adv Funct Mater. 2020. https:// doi. org/ 10. 1002/ adfm. 20200 7475. Publisher's Note Springer Nature remains neutral with regard to 58. Yang Z-y, Zhong Y-y, Zheng J, Liu Y, Li T, Hu E, Zhu X-f, Ding jurisdictional claims in published maps and institutional affiliations. R-q, Wu Y, Zhang Y, Tang T, He F, Wang S-s, Wang Y. Fmoc- amino acid-based hydrogel vehicle for delivery of amygdalin to perform neuroprotection. Smart Mater Med. 2021;2:56. 1 3 Advanced Fiber Materials Kaiyuan Yang graduated from Tsinghua University and is now a Yaosai Liu graduated from Xuzhou Medical University and is now a resident at Department of Neurosurgery, Beijing Tsinghua Chang- Ph.D. Student at the School of Clinical Medicine, Tsinghua University. gung Hospital, School of Clinical Medicine, Tsinghua University. His His research focuses on the construction of drug delivery scaffolds for research focuses on the construction of multifunctional scaffolds for spinal cord repair. spinal cord repair. Shuhui Yang graduated from Tsinghua University and she joined the Jia Yang graduated from Beijing University of Technology and is now Zhejiang Sci-Tech University and the School of Materials Science and a Ph.D. Student at the School of Materials Science and Engineering, Engineering as an Associate Professor in 2022. Her research focuses on Tsinghua University. Her research focuses on the construction of 3D the construction of multifunctional scaffolds for peripheral nerve repair. bioprinting scaffolds for spinal cord repair. Yi Guo graduated from Capital Medical University and is now an Weitao Man graduated from Tsinghua University and is now a post- Associate Professor at Department of Neurosurgery, Beijing Tsinghua doctoral fellow at Massachusetts General Hospital, Harvard University. Changgung Hospital, School of Clinical Medicine, Tsinghua Univer- His research focuses on the construction of multifunctional scaffolds sity. His research focuses on the construction of degradable biomateri- for spinal cord repair. als for brain trauma repair. Zhe Meng graduated from Capital Medical University and is now a Zhijun He is now a postgraduate student at the School of Clinical Med- Ph.D. Student at the School of Clinical Medicine, Tsinghua University. icine, Tsinghua University. He aims to develop nanoscale drug-delivery His research focuses on the construction of multifunctional scaffolds systems for use in spinal cord repair. for spinal cord repair. Chao Ma graduated from Capital Medical University and is now a Chun‑Yi Yang is now a Ph.D. Student at the School of Materials Sci- Ph.D. Student at the School of Clinical Medicine, Tsinghua University. ence and Engineering, Tsinghua University. His research interests focus His research focuses on the construction of conductive biomaterials for on the use of functional polymer materials and their processing for spinal cord repair. spinal cord repair. Guihuai Wang received his Ph.D. degree in neurosurgery from Capi- Zheng Cao graduated from Tsinghua University and is now a post- tal Medical University in 1997. He is now working as a professor at doctoral fellow at the School of Materials Science and Engineering, Department of Neurosurgery, Beijing Tsinghua Changgung Hospital, Tsinghua University. Her research focuses on the construction of mul- School of Clinical Medicine, Tsinghua University. His research inter- tifunctional scaffolds for spinal cord repair. ests include the construction of nanomaterials and scaffolds for spinal cord repair and anti-tumor therapy. Jun Liu is now a Ph.D. Student at the School of Materials Science and Engineering, Tsinghua University. His research focus on the drug Xiumei Wang received her Ph.D. degree in materials science and delivery for biomedical applications. engineering from Tsinghua University in 2005. She worked as a post- doctoral fellow at University of Rochester and Massachusetts Institute Kunkoo Kim is now a postgraduate student at the School of Materials of Technology from 2005 to 2008. She is now working as a professor Science and Engineering, Tsinghua University. His research interests at the School of Materials Science and Engineering, Tsinghua Univer- focus on the use of degradable biomaterials and their processing for sity. Her research interests include the fabrication of multifunctional biomedical applications. biomaterials for tissue engineering and regenerative medicine. 1 3
Advanced Fiber Materials – Springer Journals
Published: Mar 13, 2023
Keywords: N-cadherin; Aligned fibrin nanofiber scaffold; Niche; Neural stem cell; Spinal cord injury
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