Exosomes‐Loaded Electroconductive Hydrogel Synergistically Promotes Tissue Repair after Spinal Cord Injury via Immunoregulation and Enhancement of Myelinated Axon Growth

Advanced ScienceEarly View 2105586 Research ArticleOpen Access Exosomes-Loaded Electroconductive Hydrogel Synergistically Promotes Tissue Repair after Spinal Cord Injury via Immunoregulation and Enhancement of Myelinated Axon Growth Lei Fan, Lei Fan School of Materials Science and Engineering and National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, No. 381, Wushan Road, Tianhe District, Guangzhou, 510641 ChinaSearch for more papers by this authorCan Liu, Can Liu Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003 ChinaSearch for more papers by this authorXiuxing Chen, Xiuxing Chen Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Department of Medical Oncology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, No. 107, Yanjiang West Road, Yuexiu District, Guangzhou, Guangzhou, 510120 ChinaSearch for more papers by this authorLei Zheng, Lei Zheng Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, No. 1838, Guangzhou Avenue North, Baiyun District, Guangzhou, Guangdong, 510515 ChinaSearch for more papers by this authorYan Zou, Yan Zou Department of Radiology, the Third Affiliated Hospital of Sun Yat-sen University, No. 600, Tianhe Road, Tianhe District, Guangzhou, 510630 ChinaSearch for more papers by this authorHuiquan Wen, Huiquan Wen Department of Radiology, the Third Affiliated Hospital of Sun Yat-sen University, No. 600, Tianhe Road, Tianhe District, Guangzhou, 510630 ChinaSearch for more papers by this authorPengfei Guan, Pengfei Guan Department of Pediatric Orthopedic, Center for Orthopedic Surgery, The Third Affiliated Hospital of Southern Medical University, No.183, Zhongshan Avenue West, Guangzhou, 510515 ChinaSearch for more papers by this authorFang Lu, Fang Lu School of Preclinical Medicine, Beijing University of Chinese Medicine, No.11, North Third Ring East Road, Chaoyang District, Beijing, 100029 ChinaSearch for more papers by this authorYian Luo, Yian Luo School of Chemical Engineering and Light Industry, Guangdong University of Technology, No.100, Waihuan West Road, Panyu District, Guangzhou, 510006 ChinaSearch for more papers by this authorGuoxin Tan, Guoxin Tan School of Chemical Engineering and Light Industry, Guangdong University of Technology, No.100, Waihuan West Road, Panyu District, Guangzhou, 510006 ChinaSearch for more papers by this authorPeng Yu, Peng Yu School of Materials Science and Engineering and National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, No. 381, Wushan Road, Tianhe District, Guangzhou, 510641 ChinaSearch for more papers by this authorDafu Chen, Dafu Chen Laboratory of Bone Tissue Engineering, Beijing Research Institute of Orthopaedics and Traumatology, Beijing JiShuiTan Hospital, No.31, Xinjiekou East Street, Xicheng District, Beijing, 100035 ChinaSearch for more papers by this authorChunlin Deng, Chunlin Deng School of Materials Science and Engineering and National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, No. 381, Wushan Road, Tianhe District, Guangzhou, 510641 ChinaSearch for more papers by this authorYongjian Sun, Corresponding Author Yongjian Sun nysysyj@163.com Department of Pediatric Orthopedic, Center for Orthopedic Surgery, The Third Affiliated Hospital of Southern Medical University, No.183, Zhongshan Avenue West, Guangzhou, 510515 China E-mail: nysysyj@163.com, zhoul@gzhmu.edu.cn, imcyning@scut.edu.cn, ning_lab@hotmail.comSearch for more papers by this authorLei Zhou, Corresponding Author Lei Zhou zhoul@gzhmu.edu.cn Guangzhou Key Laboratory of Spine Disease Prevention and Treatment, Department of Spine Surgery, The Third Affiliated Hospital, Guangzhou Medical University, No. 63, Duobao Road, Liwan District, Guangzhou, 510150 China E-mail: nysysyj@163.com, zhoul@gzhmu.edu.cn, imcyning@scut.edu.cn, ning_lab@hotmail.comSearch for more papers by this authorChengyun Ning, Corresponding Author Chengyun Ning imcyning@scut.edu.cn ning_lab@hotmail.com orcid.org/0000-0003-3293-4716 School of Materials Science and Engineering and National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, No. 381, Wushan Road, Tianhe District, Guangzhou, 510641 China E-mail: nysysyj@163.com, zhoul@gzhmu.edu.cn, imcyning@scut.edu.cn, ning_lab@hotmail.comSearch for more papers by this author Lei Fan, Lei Fan School of Materials Science and Engineering and National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, No. 381, Wushan Road, Tianhe District, Guangzhou, 510641 ChinaSearch for more papers by this authorCan Liu, Can Liu Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003 ChinaSearch for more papers by this authorXiuxing Chen, Xiuxing Chen Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Department of Medical Oncology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, No. 107, Yanjiang West Road, Yuexiu District, Guangzhou, Guangzhou, 510120 ChinaSearch for more papers by this authorLei Zheng, Lei Zheng Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, No. 1838, Guangzhou Avenue North, Baiyun District, Guangzhou, Guangdong, 510515 ChinaSearch for more papers by this authorYan Zou, Yan Zou Department of Radiology, the Third Affiliated Hospital of Sun Yat-sen University, No. 600, Tianhe Road, Tianhe District, Guangzhou, 510630 ChinaSearch for more papers by this authorHuiquan Wen, Huiquan Wen Department of Radiology, the Third Affiliated Hospital of Sun Yat-sen University, No. 600, Tianhe Road, Tianhe District, Guangzhou, 510630 ChinaSearch for more papers by this authorPengfei Guan, Pengfei Guan Department of Pediatric Orthopedic, Center for Orthopedic Surgery, The Third Affiliated Hospital of Southern Medical University, No.183, Zhongshan Avenue West, Guangzhou, 510515 ChinaSearch for more papers by this authorFang Lu, Fang Lu School of Preclinical Medicine, Beijing University of Chinese Medicine, No.11, North Third Ring East Road, Chaoyang District, Beijing, 100029 ChinaSearch for more papers by this authorYian Luo, Yian Luo School of Chemical Engineering and Light Industry, Guangdong University of Technology, No.100, Waihuan West Road, Panyu District, Guangzhou, 510006 ChinaSearch for more papers by this authorGuoxin Tan, Guoxin Tan School of Chemical Engineering and Light Industry, Guangdong University of Technology, No.100, Waihuan West Road, Panyu District, Guangzhou, 510006 ChinaSearch for more papers by this authorPeng Yu, Peng Yu School of Materials Science and Engineering and National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, No. 381, Wushan Road, Tianhe District, Guangzhou, 510641 ChinaSearch for more papers by this authorDafu Chen, Dafu Chen Laboratory of Bone Tissue Engineering, Beijing Research Institute of Orthopaedics and Traumatology, Beijing JiShuiTan Hospital, No.31, Xinjiekou East Street, Xicheng District, Beijing, 100035 ChinaSearch for more papers by this authorChunlin Deng, Chunlin Deng School of Materials Science and Engineering and National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, No. 381, Wushan Road, Tianhe District, Guangzhou, 510641 ChinaSearch for more papers by this authorYongjian Sun, Corresponding Author Yongjian Sun nysysyj@163.com Department of Pediatric Orthopedic, Center for Orthopedic Surgery, The Third Affiliated Hospital of Southern Medical University, No.183, Zhongshan Avenue West, Guangzhou, 510515 China E-mail: nysysyj@163.com, zhoul@gzhmu.edu.cn, imcyning@scut.edu.cn, ning_lab@hotmail.comSearch for more papers by this authorLei Zhou, Corresponding Author Lei Zhou zhoul@gzhmu.edu.cn Guangzhou Key Laboratory of Spine Disease Prevention and Treatment, Department of Spine Surgery, The Third Affiliated Hospital, Guangzhou Medical University, No. 63, Duobao Road, Liwan District, Guangzhou, 510150 China E-mail: nysysyj@163.com, zhoul@gzhmu.edu.cn, imcyning@scut.edu.cn, ning_lab@hotmail.comSearch for more papers by this authorChengyun Ning, Corresponding Author Chengyun Ning imcyning@scut.edu.cn ning_lab@hotmail.com orcid.org/0000-0003-3293-4716 School of Materials Science and Engineering and National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, No. 381, Wushan Road, Tianhe District, Guangzhou, 510641 China E-mail: nysysyj@163.com, zhoul@gzhmu.edu.cn, imcyning@scut.edu.cn, ning_lab@hotmail.comSearch for more papers by this author First published: 06 March 2022 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Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Electroconductive hydrogels are very attractive candidates for accelerated spinal cord injury (SCI) repair because they match the electrical and mechanical properties of neural tissue. However, electroconductive hydrogel implantation can potentially aggravate inflammation, and hinder its repair efficacy. Bone marrow stem cell-derived exosomes (BMSC-exosomes) have shown immunomodulatory and tissue regeneration effects, therefore, neural tissue-like electroconductive hydrogels loaded with BMSC-exosomes are developed for the synergistic treatment of SCI. These exosomes-loaded electroconductive hydrogels modulate microglial M2 polarization via the NF-κB pathway, and synergistically enhance neuronal and oligodendrocyte differentiation of neural stem cells (NSCs) while inhibiting astrocyte differentiation, and also increase axon outgrowth via the PTEN/PI3K/AKT/mTOR pathway. Furthermore, exosomes combined electroconductive hydrogels significantly decrease the number of CD68-positive microglia, enhance local NSCs recruitment, and promote neuronal and axonal regeneration, resulting in significant functional recovery at the early stage in an SCI mouse model. Hence, the findings of this study demonstrate that the combination of electroconductive hydrogels and BMSC-exosomes is a promising therapeutic strategy for SCI repair. 1 Introduction An estimated 27 million people live with long-term disability following spinal cord injury (SCI) worldwide, with ≈180 000 new cases occurring each year.[1] SCI is followed by neuronal loss and axon and myelin necrosis, which leads to an extensive inflammatory response and further exacerbates secondary damage.[2] Meanwhile, activated inflammatory cells (microglia) release proinflammatory cytokines, which contribute to reactive astrocyte gathering, subsequently increasing the release of inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs) at the injury site.[3] Because of the inflammatory microenvironment and limited neural regeneration capacity, the injury is resolved by the formation of a dense glial scar, which acts as a barrier to neural and axonal regeneration.[4] Thus, modulation of the inflammatory microenvironment, enhancement of neural stem cell (NSCs) recruitment and neuronal regeneration, and guidance and promotion of myelinated axon growth are needed for treating spinal cord injury (SCI). Decompressive surgery with re-establishment of spinal stability and high-dose intravenous methylprednisolone sodium succinate (MPSS) usage in the acute phase of injury (≤8 h) are the most common clinical treatments at present.[5] However, the former only aims to avoid further secondary damage by relieving the pressure on the injured spinal cord, and the latter can reduce early inflammatory responses but with severe complications, while neither show the ability to promote axonal and neural regeneration and therefore, have limited therapeutic effectiveness. In this regard, experimental approaches to promote axon growth including cellular transplantation and scaffold biomaterials have been applied in SCI repair.[6] Cellular transplantation has been used experimentally in clinical conditions with some success but continues to be limited by uncontrolled cell differentiation, low survival rates, ethical issues, and the inevitable cell loss after implantation.[7] Considering the seriousness of these problems, cost-effective and cell-free biomaterial implants are highly desirable. Scaffold biomaterial-based therapy with tunable modulus, topology, patterned surfaces has been proposed as a strategy to promote neural tissue regeneration by providing 3D matrices with the desired biological, chemical, and physical characteristics that favor cellular attachment, growth, differentiation, and neurite extension.[8, 9] In particular, since the soft and hydrated forms of hydrogels are similar to native nerve tissue, they are widely used to promote cellular growth and tissue formation after SCI.[10, 11] Our previous study revealed that the mechanical properties of the hydrogel could modulate the fates of NSCs which are sensitive to mechanical changes. NSCs were inclined to differentiate into neurons and lengthen axons in soft scaffolds; otherwise, NSCs are prone to differentiate into astrocyte in slightly stiffer materials.[12, 13] However, hydrogels with poor conductivity limit their application in regulating the function of excitable cell types such as muscle and nerve cells.[11] Importantly, mimicking the electrical transmission properties of the native nerve tissue is highly beneficial for SCI repair. Currently, electroconductive hydrogels have emerged as a promising class of hydrogel scaffolds combining a hydrophilic matrix with conducting components such as electroconductive polymers, metallic nanoparticles, or carbon materials.[12] Due to its tissue-like softness and the inherent presence of electrical fields similar to the innate nervous system, the electroconductive hydrogel can provide mechanical and electrical cues for enhancing neuronal differentiation of NSCs and controlling neurite extension.[11, 13] We previously developed a porous, highly electroconductive, soft, and biocompatible conducting polymer hydrogel, which forms a freestanding electroconductive hydrogel for implantation into the spinal cord hemisection gap, and recently demonstrated that implanting this electroconductive hydrogel after SCI stimulated endogenous NSCs recruitment and neuronal differentiation after SCI.[14-16] Although electroconductive hydrogels effectively enhanced neuronal and axonal regeneration, their efficacy is compromised by host recognition and the subsequent foreign body immune responses, which cannot attenuate or even aggravate the early secondary inflammation after acute SCI.[17, 18] Thus, single transplantation of electroconductive hydrogel may not be sufficient to achieve a substantial improvement in SCI repair. Bone marrow stem cell (BMSC) therapy shows characteristic immunomodulatory effects and has been applied in severe clinical inflammatory diseases such as pancreatitis, colitis, and focal cerebral ischemia.[15] Exosomes are involved in intercellular communication and act on the innate immune system as paracrine messengers. They also exert immunomodulatory effects and can alleviate immune abnormalities.[19, 20] Recently, BMSC-exosomes have emerged as a new cell-free therapeutic platform for various diseases due to their therapeutic effects, including their ability to promote regeneration and modulate immunoreaction.[21] BMSC-exosomes, as a promising nanocarriers, carry various therapeutic growth factors and miRNAs, leading to axonal regeneration and angiogenesis enhancement, structural and electrophysiological improvements, and neuroinflammation and gliosis reduction, resulting in significant motor improvement and sensory recovery after spinal cord injury.[21, 22] Meanwhile, neuroinflammation reduction caused by miRNAs contained in BMSC-exosomes can negatively regulate the TLR4/NF-κB pathway.[23, 24] Delivery of immune-modulating BMSC-exosomes in electroconductive hydrogel can attenuate adverse host immune response and exert synergistically therapeutic effects in combination with electroconductive hydrogel to promote functional recovery. However, exosome-loaded electroconductive hydrogel systems have not been investigated for their ability to promote tissue repair. In view of this, we hypothesized that an electroconductive hydrogel combined with BMSC-exosomes might achieve adequate therapeutic effectiveness in patients with SCI. Moreover, the delivery of BMSC-exosomes in electroconductive hydrogels can attenuate adverse host immune effects while synergistically exerting the therapeutic effect of promoting neuronal and axonal regeneration, thereby alleviating SCI. Herein, we developed exosome-loaded dual-network electroconductive hydrogels composed of photo-cross-linkable gelatin methacrylate (GM) hydrogels and polypyrrole (PPy) hydrogels. First, the GM/PPy (GMP) hydrogel scaffold was fabricated by in situ growth of the PPy network cross-linked and doped by tannic acid (TA) within the GM hydrogel networks, which possesses natural cell binding motifs such as Arg–Gly–Asp (RGD), allowing cells to grow within it. Then, BMSC-exosomes were immobilized in the TA-doped GMP hydrogel network to form a GM/PPy/exosomes (GMPE) hydrogel via reversible interactions formation due to the presence of large amounts of polyphenol groups in TA. The noncovalent binding did not affect the structure and bioactivity of the exosomes while ensuring a slow sustained release of exosomes early in the implantation. Cell biocompatibility, adhesion, growth, and differentiation on the GMPE hydrogel scaffold were evaluated in vitro. In addition, the specific signaling pathways via which GMPE hydrogels manipulate immune response and promote axonal regeneration were identified. A mouse spinal cord hemisection model was established to detect whether the GMPE hydrogel was efficient in facilitating nerve regeneration and improving functional recovery after SCI. 2 Results 2.1 Identification of BMSCs and BMSC-Exosomes Since nanoparticle tracking analysis (NTA) assay is a well-established technique to quantify the concentration of exosomes according to MISEV2018 guidelines,[25] we have conducted NTA assay to confirm that exosome-free fatal bovine serum (FBS) successfully obtained by ultracentrifugation (Figure S1A, Supporting Information). The exosome-free media was used as the cell cultures to isolate BMSC-exosomes. The extraction of BMSCs and BMSC-exosomes is illustrated in Figure S1B of the Supporting Information. Photomicrographs showed that primary BMSCs typically exhibited spindle-like morphology (Figure S1C, Supporting Information). Flow cytometry showed that the obtained cells expressed high levels of the positive BMSCs surface markers such as CD29, CD90, and CD44H, but did not express negative surface markers such as CD11b and CD45 of BMSCs (Figure S1C, Supporting Information). These cells also successfully underwent adipogenic, osteogenic, and chondrogenic differentiation (Figure S1D, Supporting Information), which indicated the successful extraction of BMSCs. The presence of a cup-shaped morphology was observed with transmission electron microscopy (TEM) analysis (Figure S1E, Supporting Information), a particle size of 70 to 140 nm from NTA devices (Figure S1F, Supporting Information), and the expression of Flotillin-1, CD63, and TSG101 on the nanoparticles surface (Figure S1G, Supporting Information) all demonstrated successful extraction of BMSC-derived exosomes. We have performed proteomic mass spectrometry to analyze the purity of the collected exosomes on the basis of the standards suggested by the International Society for Extracellular Vesicles (ISEV).[19] The result showed that the absence of cellular or serum contaminants (Table S1, Supporting Information). 2.2 Fabrication of GM, GMP, and GMPE Hydrogels We used a three-step synthesis process to produce GMPE hydrogels (Figure 1A,B). First, the GM hydrogel networks were formed by ultraviolet (UV) light photo-cross-linking of GM units. Secondly, the GM hydrogel was successively immersed into solution I containing the monomers Py and TA and solution II containing ammonium persulfate (APS), allowing in situ polymerization and cross-linking of conducting PPy chains. Transparent GM hydrogels become opaque with the formation of the PPy (black color), demonstrating successful polymerization of polypyrrole in the GM hydrogel (Figure S2A,B, Supporting Information). TA is an abundant natural water-soluble polyphenol present in plants that can act as a dopant and cross-linker for PPy hydrogel formation.[11] In this study, TA interacted with the amide bond on the GM backbone via hydrogen bonds and also reacted with PPy chains by protonating the nitrogen groups on PPy to form a dual-network electroconductive hydrogel (GMP hydrogel) with strong interactions. This GM network conferred biocompatibility, tissue-like softness, degradability, and tissue and cell adhesion, while the PPy network provided electroconductive electrical activity to the hydrogels. Finally, the BMSC-exosomes were immobilized into the GMP hydrogel network to form the GMPE hydrogel via reversible reversible interactions formation between the presence of a large amount of polyphenol groups in TA and the phosphate groups in the phospholipid of exosomes. Figure 1Open in figure viewerPowerPoint Characteristics of the GMPE hydrogels. A) Illustration of how the GMPE hydrogel can reduce early inflammation, enhance NSCs recruitment and promote myelin-associated axonal regrowth to synergistically promote locomotor recovery after spinal cord hemisection. B) The three-step synthesis procedure for the GMPE hydrogel was illustrated. The GMP hydrogel was synthesized by TA interacting with the amide bond on the GM backbone and the nitrogen groups on PPy chains. BMSC-exosomes were reversibly immobilized into GMP hydrogels via hydrogen bond formation between TA polyphenol groups and phosphate groups in exosomes phospholipid to form GMPE hydrogel. C) Microstructure of the GMPE hydrogel was observed by SEM. Scale bars: 25 µm. D) Electrical characterization, including CV, EIS, I–V, and Bode plots of GMP and GMPE hydrogels showed excellent electrical performance. E) After the isolation of transected spinal cords, the stimulating electrical signals were retransmitted by GMPE hydrogels. F) IF imaging showed that exosomes were evenly distributed into the GMPE hydrogel and the penetration depth of the exosomes was more than 100 µm. Scale bars: 100 µm. G) RT-qPCR indicated that BMSC-exosomes express of axonal regeneration-related, remyelination-related, and anti-inflammatory-related miRNAs (n = 3). H) BV2 cells cultured on the GMPE hydrogel can normally phagocytize exosomes released from the hydrogel. White arrows indicate where BV2 cells have phagocytosed exosomes. Scale bars: 100 µm. I) Anti-inflammatory-related miRNAs expression increased as the result of BV2 cells phagocytosing exosomes (n = 3). J) PKH26-labeled exosomes were clearly detected in the cytoplasm of NSCs, suggesting successful in vitro endocytosis of exosomes released from the GMPE hydrogel. White arrows indicate where NSCs have phagocytosed exosomes. Scale bars: 100 µm. K) Axonal regeneration-related and remyelination-related miRNAs expression increased after NSCs phagocytize exosomes (n = 3). 2.3 Characteristics of GM, GMP, and GMPE Hydrogel In comparison with gelatin, GM showed two new peaks at 5.3 and 5.5 ppm that were attributed to the two protons of its methacrylate groups (Figure S2C, Supporting Information). The GM hydrogel exhibited amide bands characteristic of gelatin, including C═O stretching at 1650 cm−1 (amide I), CN at 1440 cm−1, and NH deformation at 1239 cm−1 (amide III). In the PPy spectrum, the peaks at 1556 and 1403 cm−1 are Py ring vibrations. These peaks also appeared in the spectra of the GMP hydrogels, indicating that the PPy chain was successfully incorporated into the GM hydrogel backbone (Figure S2D, Supporting Information). Scanning electron microscopy (SEM) analysis showed that GMPE hydrogels exhibited a 3D highly porous structure (Figure 1C, Supporting Information), which provided space for nerve cell extension. In addition, high-magnification SEM also showed the interconnected globular nanoparticle morphology of PPy was coated onto the GM backbone (Figure S2E, Supporting Information). The mechanical properties of all samples were tested using dynamic oscillatory frequency sweep measurements. The storage moduli (elastic modulus, G′) of all hydrogels were larger than the loss moduli (viscous modulus, G″) over an angular frequency range of 1–100 Hz, indicating that the hydrogels had good stability (Figure S2F, Supporting Information). The average storage modulus at a 10 Hz angular frequency increased from 555.7 ± 50.1 Pa for the GM hydrogel to 1039.3 ± 89.3 Pa and 1056.0 ±133.1 Pa for the GMP and GMPE hydrogels, respectively (Figure S2G, Supporting Information). However, the mechanical properties of all three hydrogels matched neural tissue mechanics (600–3000 Pa), which was beneficial for cell function and differentiation. The introduction of hydrophobic PPy reduced the swelling ratio in the GMPE and GMP hydrogels when compared with GM hydrogel, although the difference was not statistically significant (Figure S2H, Supporting Information). Also, introducing TA may also contribute the mechanical strength enhancement and the swelling ratio reduction because of the strong interactions between polymers and the phenol groups in the TA molecules.[26] Additionally, after soaking the hydrogel in the physiological medium for 7 and 14 days, the swelling ratio and mechanical properties of the GMPE hydrogel did not change significantly (Figure S2I–J, Supporting Information), indicating that the hydrogel exhibited long-term swelling effect and mechanical stability. Ex vivo spinal cords were able to stick to the GMPE hydrogel, which indicated that the hydrogels also had excellent bioadhesion and supported their potential use in an in vivo animal SCI model (Figure S2K, Supporting Information). The plateau value of force/width was ≈2.4 N m−1 during the peeling adhesion test, comparable to that of clinically used fibrin glue. (Figure S2L, Supporting Information). To probe the electrochemical properties of GMP and GMPE hydrogels, a hydrogel electrode was prepared by in situ gelation of electroconductive hydrogels onto a piece of indium-tin oxide (ITO). cyclic voltammetry (CV), and electrochemical impedance (EIS) were performed with 0.1 m phosphate buffered saline (PBS, pH 7.4) as the electrolyte. Compared to the GM hydrogel, the GMP and GMPE hydrogels showed significantly improved anodic and cathodic currents (Figure 1D). The CV curves showed similar oxidation and reduction current values for GMP and GMPE hydrogels. The EIS imaging showed a quasi-semicircle in the high-frequency region of the Nyquist plots of the GMP and GMPE hydrogels, indicating that they exhibited good redox activity. Additionally, the diameter of this quasi-semicircle was related to the charge transfer resistance. The larger the radius of the circle, the larger the charge transfer resistance. The diameter of the semicircle for the GM hydrogel was significantly larger than that for the GMPE and GMP hydrogels, which indicated that the GMP and GMPE hydrogels both exhibited better electrical performance in comparison with GM hydrogels. In addition, the current–voltage (I–V) curves showed that the conductivities of GMP and GMPE hydrogels were 1.83 × 10−3 and 1.49 × 10−3 S cm−1, respectively, which were significantly higher than those of the GM hydrogel. As the Bode plots show, both GMP and GMPE hydrogels showed significantly low impedance values that were between 300 Hz and 1 kHz compared with GM hydrogel. These values are within the frequency of the exchange signals observed in nerve cells.[27] Together, these data show that GMP and GMPE hydrogels exhibited similar electrical properties to each other, indicating that the introduction of exosomes had no obvious effect on the electrical properties of the electroconductive hydrogels. An isolated spinal cord circuit test was used to further evaluate the ability of the hydrogel to transmit electrical signals. After the ex vivo mouse spinal cord was transected, no electrical signal transmission was recorded below the injury site (Figure 1E). When two ends of injury site were bridged by GMPE hydrogel, the stimulating electrical signals were able to be transmitted (Figure 1E), which indicated that the GPME hydrogel could partly restore endogenous electrical signaling transmission. In addition, the GMPE hydrogel also exhibited electrical stability in physiological medium for more than 2 weeks (Figure S2M, Supporting Information), which shows that it has potential property for long-term in vivo use. The retention of BMSC-exosomes in GMPE hydrogel were investigated to evaluate the delivery capacity of the hydrogels. To visualize exosomes loaded on hydrogel, exosomes was labeled by PKH26 dyes and imaged via confocal microscopy. The 3D immunofluorescence (IF) imaging showed that in addition to a small amount of exosomes aggrega