Treatment of Denervated Muscle Atrophy by Injectable Dual-responsive Hydrogels Loaded with Extracellular Vesicles

Treatment of Denervated Muscle Atrophy by Injectable Dual-responsive Hydrogels Loaded with Extracellular Vesicles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Treatment of Denervated Muscle Atrophy by Injectable Dual-responsive Hydrogels Loaded with Extracellular Vesicles Jianzhong Du, Ziheng Bu, Jianxing Jing, Wei Liu, Zhen Fan, Junchao Huang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4715142/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Denervated muscle atrophy is a common complication following nerve injury, which often leads to irreversible muscle fibrosis due to low treatment efficiency. Recently, bioactive substances such as extracellular vesicles (EVs) have been emerging as an effective therapeutic modality for muscle atrophy. However, the complicated microenvironments of denervated muscle atrophy could reduce the delivery efficiency and even result in the deactivation of EVs. To meet this challenge, an ultrasound and pH-responsive anti-inflammatory injectable hydrogel was developed, which can effectively load and deliver stem cells derived EVs with satisfactory treatment outcomes of denervated muscle atrophy. Carboxymethyl chitosan, oxidized chondrotin sulfate and cystamine dihydrochloride were crosslinked in situ by Schiff base reaction to form an injectable hydrogel, where the reversible covalent bond would break under ultrasound and acidic environments to promote hydrogel degradation and cargo release. Meanwhile, the hydrogel loaded with EVs isolated from human umbilial cord mesenchymal stem cells(HUC-MSCs) can release EVs in a controlled manner upon facile pH/ultrasound manipulation. The experimental results confirmed that the hydrogel loaded with EVs (EVs@UR-gel) was effective in preserving muscle function. After six weeks nerve reconstruction, the maximum muscle strength which is closely related to muscle function, the muscle circumference, the wet weight, can be restored to 89.53 ± 0.96%, 76.02 ± 7.49%, 88.0 ± 2.65% of the healthy state, and the sciatic nerve index (SFI) to -0.11 ± 0.09, respectively. Overall, this hydrogel provided a new platform to maintain the long-term in vivo bioactivity of EVs, achieve tunable EVs release at the site of denervated muscle atrophy based on the state of disease, and restore the morphology and function of muscle as a promising approach for treating denervated muscle atrophy. Physical sciences/Materials science/Biomaterials/Biomedical materials Biological sciences/Stem cells/Regeneration Biological sciences/Biotechnology/Biomaterials/Biomedical materials Physical sciences/Chemistry/Polymer chemistry/Biopolymers Denervated muscle atrophy extracellular vesicles Hydrogel Ultrasound Responsiveness Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Skeletal muscles are essential for human motion, executing various postures and generating and ceasing actions 1 , 2 . Muscle atrophy from nerve damage is prevalent and linked to disrupted nerve supply, contributing significantly to global disability and economic burden 3 , 4 . Early nerve repair is beneficial, but factors like immature surgical conditions and slow nerve regeneration can lead to muscle atrophy 5 , 6 . Current treatments focus on neural repair but lack strategies for target muscle 7 – 10 . Conventional clinical methods such as passive skeletal muscle exercises and electrical stimulation of atrophied muscles fail to meet the needs of both patients and clinical practitioners 11 – 13 . Denervated muscles will undergo a staged, highly programmed and complex pathological process, which may lead to irreversible atrophy and lifelong motor dysfunction if it exceeds the self-healing ability 14 – 20 . However, current treatments simply target specific stages of the disease, lacking a comprehensive approach to inhibit denervated muscle atrophy from multiple perspectives. Mesenchymal stem cell (MSC) therapy has shown potential in treating muscle atrophy, but it faces challenges related to cell source, tissue origin, and cellular heterogeneity 21 – 26 . EVs derived from stem cells offer comparable benefits without the issues associated with live cell use 27 , 28 . Moreover, EVs contain miRNAs, ncRNAs and growth factors, playing an important role in cell communication and regulating cell function 29 – 31 . Initial research indicates that these contents are enriched in pathways related to cellular metabolism and may counteract atrophic changes, with their reparative effects being dose-dependent 32 – 37 . Our preliminary research also indicates that treating bone and cartilage injuries with varying concentrations of EVs results in better treatment outcomes with higher concentrations significantly outperforming lower concentrations 30 , 38 . However, the in vivo stability and bioavailability of EVs are compromised, necessitating encapsulation in hydrogels for tunable release and sustained bioactivity 39 – 43 . Therefore, the development of EVs capable of early anti-inflammatory and antioxidant action, along with sustained "burst release" of EVs at specific stages for the purpose of programmed phased therapeutic strategies, is essential for the clinical application of EVs therapy. Bioengineering has shown promise in treating musculoskeletal injuries, but tailored material requirements for muscle repair, which demands high compliance and fatigue resistance, are still under investigation 44 – 46 . The initial research findings indicate that injectable hydrogels can respond to repeated mechanical stress through a process known as mechanochemical conversion. This enables external intervention to induce the transition of injectable hydrogels from a sol state to a gel state, which in turn facilitates the filling, curing, and drug release processes 47 , 48 . In the context of denervated muscle atrophy, the distinctive pathophysiology and evident staging of the condition necessitate the development of biomaterials that are injectable, possess anti-inflammatory and antioxidative properties, and are capable of dynamically and precisely regulating EVs release based on real-time therapeutic efficacy assessment during specific time intervals. In light of this concept and the pressing clinical demands, we demonstrated that umbilical cord mesenchymal stem cells-derived EVs (HUC-MSCs-EVs) effectively promote the migration, proliferation, and differentiation of myoblasts. Then we prepared a pH/ultrasound dual-responsive hydrogel (UR-gel) as the EVs carrier, achieving early anti-inflammatory and antioxidative effects at the lesion site through pH response. Additionally, based on the therapeutic efficacy, we utilized ultrasound at specific stages to achieve instantaneous burst release of HUC-MSCs-EVs, combating muscle atrophy and maintaining muscle function and morphology. This demonstrates the effectiveness of treating denervated muscle atrophy by addressing the disease's pathophysiological processes and utilizing bioactive materials. It offers new avenues and methods for clinical denervated muscle atrophy treatment. This comprehensive therapeutic approach combines the characteristics of biomaterials with the bioactivity of HUC-MSCs-EVs, with the potential to improve the quality of life for patients suffering from denervated muscle atrophy and to slow disease progression. Further research and clinical practice may lead to this treatment method becoming a significant therapeutic intervention for denervated muscle atrophy. Results and Discussion 2.1 Synthesis and Characterizations of UR-gel The synthesis of UR-gel was illustrated in scheme 1. Oxidized chondroitin sulfate (OCS) was first obtained from chondroitin sulfate (CS) by NaIO 4 oxidation. As shown in the Fig. 1 a, the stretching vibration band of aldehyde group at 1736.5 cm − 1 appeared in the FTIR spectrum of the OCS, demonstrating successful oxidation. Then, the hydrogel was synthesized by mixing OCS, carboxymethyl chitosan (CMCS) and cystamine dihydrochloride solutions. The hydrogel was spontaneously crosslinked in situ through the Schiff base reaction between the amino and aldehyde groups. The FTIR spectrum of the UR-gel showed the disappearance of the characteristic peak at 1736.5 cm − 1 , which demonstrated that the aldehyde groups of OCS were fully reacted by the amino groups of CMCS as well as cystamine dihydrochloride, and the successful crosslinking of UR-gel. Obvious porous network was observed in SEM image of hydrogel as shown in Fig. 1 b, which could be used to encapsulate EVs within the cavity. In addition, we observed the morphology of the UR-gel after ultrasound stimulation. As shown in Fig. 1 c, the pore sizes of the UR-gel increased after ultrasound stimulation. This is because the ultrasound stimulation partially destroyed the cross-linking structure of the UR-gel 49 , 50 . Meanwhile, the rheological properties of the hydrogels were measured before and after ultrasound stimulation. As shown in the Fig. 1 d, the storage modulus ( G ′) is greater than loss modulus ( G ′′) at a strain of 0.1%. With increasing strain to 1002.14%, G ′ is smaller than G ′′, suggesting that UR-gel structure was destroyed. Furthermore, G ′ of hydrogel after ultrasound stimulation was lower than that before ultrasound stimulation, indicating that ultrasound could reduce its storage modulus and accelerate the destroying of hydrogels. As shown in Fig. 1 e, swelling study revealed that the UR-gel could absorb large amount of water, and the water absorption rate maintained at about 50 times after 8 h and onwards, which indicated that UR-gel had great swelling performance and drug storage capacity. We also investigated the biodegradability with UR-gel immersing in PBS buffer containing 0.4 mg/mL lysozyme. The degradation rate increased with increasing time and reached 69.92% after immersing 6 d (Fig. 1 f). Furthermore, since the UR-gel was cross-linked by reversible covalent bonds, the UR-gel had the ability to realize the injection capacity as shown in Fig. 1 g 51 . In addition, we investigated the drug release behavior of UR-gel using doxorubicin as the model drug (Fig. 1 h). The fluorescence under an ultraviolet light (365 nm) was observed, as shown in Fig. 1 i. It showed that model drug could load into UR-gel. The drug release rate was investigated using methylene blue as model drug. The rate was calculated based on the standardized fitting curve of a range of concentrations of methylene blue solutions (Fig. S1 a-b). As shown in Fig. 1 j, compared to the release percentage of free releasing, the drug release percentage from the hydrogel increased under ultrasound stimulation or acidic environment (pH 6.0) conditions. Moreover, the ultrasound stimulation and acidic conditions could synergistically promote the drug release percentage, which reach 92.35% release percentage after 420 min incubation. 2.2 Characteristics of HUC-MSCs-EVs HUC-MSCs and HUC-MSCs-EVs were isolated and identified. Under light microscopy, typical spindle-shaped morphology of HUC-MSCs was observed (Fig. S2a). The differentiation potential of HUC-MSCs into cartilage, bone, and fat was evaluated using Alcian blue, Alizarin Red, and Oil Red O staining, respectively (Fig. S2b). Furthermore, flow cytometry was employed to detect surface markers CD105, CD73, CD90, CD45, CD34, and HLA-DR on HUC-MSCs (Fig. S2c). The results demonstrated compliance with the identification criteria for stem cells. Subsequently, EVs were successfully isolated and extracted, and typical cup-shaped structures of EVs were observed under TEM (Fig. 2 a). NTA results showed that the diameters of vesicles are mainly in the range of 80–200 nm, with an average diameter of 142.5 nm (Fig. 2 b). WB analysis revealed high expression of EVs surface markers CD63, CD81, and CD9, while they were absent on cells (Fig. 2 c). Overall, these results confirm successful isolation of EVs from HUC-MSCs. Subsequently, sequencing and analysis of miRNA expression levels in HUC-MSCs-EVs were performed and the results revealed that abundant miRNAs are contained in HUC-MSC-EVs (Fig. 2 d). It is well known that miRNAs regulate target genes by binding to the 3’UTR of target genes or specific sequences of target genes. Multiple databases were used to predict the target genes of miRNAs enriched in HUC-MSCs-EVs (Fig. S3a). GO analysis and KEGG analysis on these target genes showed that target genes of miRNAs abundant in HUC-MSCs-EVs were enriched in pathways related to cell cycle, DNA replication, insulin resistance, and various signaling pathways, indicating their roles in cell migration, proliferation, and differentiation of C2C12 cells (Fig. S3b-c). Furthermore, the effects of HUC-MSCs-EVs on the migration, proliferation, and differentiation of C2C12 cells were validated. Initially, HUC-MSCs-EVs were labeled by using the PKH-26 staining kit and co-cultured with C2C12 cells for 24 h. As shown in Fig. 2 e, red fluorescent-labeled EVs were internalized by C2C12 cells, with the cell nuclei stained blue using DAPI and the cell cytoskeleton stained green using FITC-labeled phalloidin. It was observed that the HUC-MSCs-EVs were successfully endocytosed by C2C12 cells, primarily accumulating around the cell nuclei. Transwell migration assays demonstrated a significant enhancement in the migratory capacity of C2C12 cells upon addition of HUC-MSCs-EVs. Moreover, the control groups with varying concentrations of EVs were established to determine the impact on cells. These were designated as EVs1 (5×10 8 particles/mL) and EVs2 (1×10 9 particles/mL). The results showed that EVs2 significantly enhanced cell migration compared to EVs1 (Fig. 2 f-g), indicating that the concentration is an important factor influencing the therapeutic efficacy of HUC-MSCs-EVs. Edu staining, which reflects cell proliferation and activity, revealed a substantial increase in the number of proliferating cells in both EVs1 and EVs2 groups compared to the control. EVs2 demonstrated a notably higher proliferation rate than EVs1 (Fig. 2 h), confirming the ability of HUC-MSCs-EVs to promote C2C12 cell proliferation, with higher concentrations showing superior effects. The CCK8 assay (Fig. 2 i) corroborated the previous findings, further validating the conclusion. MYOG, a key marker in myogenic differentiation, showed that the addition of extracellular vesicles positively affected the differentiation of C2C12 cells. There was a positive correlation between vesicle concentration and the extent of myogenic differentiation, as evidenced by immunofluorescence results (Fig. 2 j). This correlation underscores the sequencing and prediction of HUC-MSCs-EVs, highlighting their potential therapeutic impact on denervated muscle atrophy. 2.3 In Vitro Biocompatibility of EVs@UR-gel To validate the in vitro biocompatibility of EVs@UR-gel, a concentration of 1×10 10 particles/ml hydrogel was prepared by adding 10 µL of 1×10 11 particles/mL into 90 µL of UR-gel. The UR-gel alone acted as the control, while UR-gel stimulated with ultrasound at an intensity of 1 MHz for 10 minutes and EVs@UR-gel the same stimulated served as experimental groups. After co-culturing with C2C12 cells for 3 days, Actin/DAPI staining results showed (Fig. 3 a) that the cells were well spread on the hydrogel scaffold. Following ultrasound stimulation, there was a noticeable increase in cell number and growth depth, which was further enhanced with the addition of EVs. CCK-8 results (Fig. 3 b) and quantitative analysis (Fig. 3 c-d) demonstrated an enhanced proliferative capacity of the material post-ultrasound treatment, which was further augmented with the addition of EVs. Quantitatively, compared to the gel group and ultrasound-stimulated gel group, the ultrasound-stimulated EVs@UR-gel group showed an increase in nuclear count from 287 ± 36 cells/field to 891 ± 31 cells/field and an increase in cell growth depth into the hydrogel from 90.5 ± 10 nm to 169.3 ± 12.7 nm. Subsequently, the effects of EVs@UR-gel on C2C12 cell migration and differentiation were verified. Figure 3 e illustrates that cell migration was promoted by UR-gel after ultrasound treatment, with an even more pronounced effect observed with EVs@UR-gel. Quantitative analysis (Fig. 3 f) confirmed that the EVs@UR-gel group had a significantly stronger effect on cell migration than both the UR-gel and ultrasound-treated UR-gel groups. Differentiation experiments, as shown in Fig. 3 g and Fig. S4, demonstrated that MyoG immunofluorescence staining indicated a marked ability of EVs@UR-gel with ultrasound treatment to enhance C2C12 cell differentiation. These results suggest that ultrasound stimulation induces structural changes in UR-gel, increasing its porosity and providing more space for C2C12 cell growth. The release of encapsulated EVs post-ultrasound stimulation aids in cell growth into deeper regions of the hydrogel. It is highlighted that the difference between the UR-gel and EVs@UR-gel groups underscores the protective role of the hydrogel on EVs, indicating that even after 10 minutes of 1 MHz ultrasound stimulation, the biological activity of EVs is preserved. 2.4 Anti-inflammatory and Antioxidant Effects of EVs@UR-gel The efficacy of EVs@UR-gel in protecting target muscles from oxidative stress and inflammation following denervation has been validated. At the early stages of denervated muscle atrophy, muscles experience a loss of voluntary contraction and fibrillation abilities, leading to blood accumulation and a hypoxic microenvironment. This hypoxia, over time, triggers inflammatory cell aggregation and a vicious cycle that accelerates muscle atrophy. The capacity of carrier materials to neutralize free radicals is therefore essential. The anti-inflammatory and antioxidant properties of EVs@UR-gel were tested through protective, ROS scavenging, and macrophage polarization experiments on C2C12 cells using UR-gel. The anti-inflammatory and antioxidant properties of EVs@UR-gel were tested through protective, ROS scavenging, and macrophage polarization experiments on C2C12 cells using UR-gel. Calcein-AM/PI staining post-H 2 O 2 treatment showed an increase in live cells with UR-gel addition, and a slight increase post-ultrasound treatment (Fig. 4 a and Fig. S5a). CCK-8 results (Fig. 4 b) indicated higher cell viability with UR-gel, regardless of ultrasound treatment, suggesting a protective effect against ROS without significant impact from ultrasound on ROS resistance. Furthermore, 2',7'-dichlorofluorescin diacetate (DCFH-DA) was used to measure intracellular ROS levels, revealing that UR-gel reduced fluorescence intensity, indicating a downregulation of ROS induced by H 2 O 2 (Fig. 4 c-d). The ability of cells to resist ROS is enhanced after ultrasound treatment, likely due to structural changes in the hydrogel after ultrasound, leading to internalization of some hydrogel components by cells and thus enhancing intracellular anti-ROS capacity. Considering the association between inflammatory response and M1 macrophage polarization, the effect of materials on anti-inflammatory activity against M1 macrophages was investigated by co-incubating with RAW264.7 cells stimulated with LPS. A significant reduction in pseudopod formation indicated a decrease in the amount of cells polarized to M1 macrophages (Fig. 4 e and Fig. S5b). Combined with the above results, the application of ultrasound significantly reduced intracellular reactive oxygen species content, restored the characteristics of M0 macrophages, and induced a rounded or ellipsoidal morphology with strong adhesion to the wall, as well as elongated spindle shape, a characteristic morphology of M2 polarization. After fluorescence labeling with anti-CD86, flow cytometry results showed that the proportions of M1 macrophages were 81.2%, 63.3%, and 51.4% in the different treatment groups, respectively, confirming the hydrogel's antioxidant and anti-inflammatory effects (Fig. 4 f). The material's strong anti-inflammatory and antioxidant capabilities are suitable for early treatment strategies of denervated muscle atrophy. Figure 4 g illustrates the immunomodulation mechanism of UR-gel. 2.5 The Therapeutic Effects of EVs@UR-gel on Motor Function Recovery in Rats To assess the reparative effect of EVs@UR-gel on denervated muscle atrophy, during model establishment, the hydrogel was injected into the tibialis anterior muscle of rats. Gross specimens showed that the tibialis anterior muscle in the EVs@UR-gel group was significantly thicker compared to other groups (Fig. 5 a). After 6 weeks of denervation, measurements indicated that the EVs@UR-gel group had a higher residual muscle circumference, retained passive muscle force, and greater muscle wet weight compared to other groups (Fig. 5 b-d). At 6 weeks post nerve repair (12th week), the maximum muscle strength which is closely related to muscle function, the muscle circumference and the wet weight, can be restored to 89.53 ± 0.96%, 76.02 ± 7.49% and 88.0 ± 2.65% of the healthy state, respectively. The intervention with EVs@UR-gel reduced the rate of muscle atrophy and loss of muscle strength post-denervation while preserving muscle recovery capability. To explore the protective effect of EVs@UR-gel on the motor ability of rats after denervation and repair, gait analysis was conducted on rats at the 12th week. Motor function was assessed using the Sciatic Functional Index (SFI), with 0 indicating normal function and − 100 indicating complete loss of motor function. In the assessment of normal motor function, values of PLF, TSF, and ITF were 0. Results indicated that at the 12th week, except for the sham surgery group, all indicators and the comprehensive index SFI in the EVs@UR-gel group were significantly higher than in the other groups (Fig. 5 e-i). These findings suggest that the designed EVs@UR-gel hydrogel can maintain the motor ability of target muscles after denervation. 2.6 Immunofluorescence Staining Results and Gross Specimens Tissue sections of the tibialis anterior muscle at the 6th week were subjected to Masson's staining and immunofluorescence staining to validate the anatomical protection and maintenance of denervated muscles by EVs@UR-gel. Masson's staining indicated that muscle fibers in the EVs@UR-gel group preserved their integrity, had a tight arrangement, and featured reduced collagen fiber thickness and an organized extracellular matrix, contrasting with the other three groups (Fig. 6 a). The extent of muscle atrophy was notably reduced, and fibrosis was less pronounced in the EVs@UR-gel group. Further immunofluorescence staining of the sections with MyHC and Col-1 antibodies showed that MyHC fluorescence intensity and morphology were significantly better in the EVs@UR-gel group compared to the other groups. The fluorescence intensity of MyHC increased sequentially from the control group to the UR-gel group, ultrasound-treated UR-gel group, and EVs@UR-gel group, while Col-1 fluorescence decreased. Subsequently, the result was subjected to further clarification through the implementation of quantitative calculations (Fig. 6 b-c). Tissue-level observations suggest that the early anti-inflammatory effects of EVs@UR-gel are advantageous in mitigating muscle atrophy and fibrosis, with ultrasound-stimulated UR-gel showing enhanced reparative effects. The release of EVs in vivo , as indicated by the EVs@UR-gel group results, promotes the preservation of target muscle function and morphology. Changes in MyHC, the principal contractile protein, and Col-1, a key component in muscle fibrosis, underscore the protective and maintenance effects of EVs@UR-gel on the function of denervated skeletal muscle. The trend of these changes also supports the correctness of our material design theory. Subsequently, mRNA sequencing was performed on the control group and EVs@UR-gel group, revealing 560 upregulated genes and 315 downregulated genes (Fig. S6). GO and KEGG analysis of these differentially expressed genes (Fig. 6 d-g) showed significant enrichment in signaling pathways related to muscle tissue development, muscle contraction, anti-inflammatory responses, and metabolic promotion. These findings imply that the application of EVs@UR-gel is capable of sustaining the function of denervated target muscles and stabilizing the internal environment at a genetic level. Methods 3.1 Synthesis of Oxidized Chondroitin Sulfate (OCS) OCS was synthesized by an oxidation reaction using NaIO 4 . First, 1.0 g of chondroitin sulfate (CS) was dissolved in 20 mL distilled water. Then, 700 mg of NaIO 4 was dissolved in 20 mL distilled water. After dissolving completely, two solutions were mixed and stirred for 6 h in the dark at room temperature. Subsequently, 1 mL of ethylene glycol was added into the reaction system to terminate the oxidation reaction for 1 h. And then, the solution was dialyzed with distilled water for 3 days and freeze-dried, resulting in a white solid sample. OCS was stored at room temperature and protected away from light. 3.2 Fabrication of pH/Ultrasound Dual-Responsive Hydrogel The pH/ultrasound dual-responsive hydrogel was prepared based on the principle of the Schiff base reaction. In brief, carboxymethyl chitosan (CMCS, 1.0 g) was dissolved in 50 mL of deionized water with stirring to completely dissolved. OCS (500 mg) was dissolved in 5 mL of deionized water to obtain 100 mg/mL of OCS. Cystamine dihydrochloride was dissolved in deionized water to 1.0 mol/L. And then, CMCS, OCS and cystamine dihydrochloride solutions were mixed at a volume ratio of 8:1:1 at room temperature with slight stirring. The pH/ultrasound dual-responsive hydrogel was gradually solidified and fabricated. 3.3 Fourier Transform Infrared Spectra (FTIR) Measurement FTIR data were recorded using a Bruker Equinox 55 spectrometer at frequencies ranging from 400 cm − 1 to 4000 cm − 1 and resolution of 0.5 cm – 1 . Samples were powdered and mixed with dried KBr powder and pressed into pellet form. 3.4 Morphology of Hydrogels The morphologies of hydrogels were analyzed by scanning electron microscopy (SEM, Zeiss microscope). Samples were washed for three time with deionized water and frozen in -20 ℃. Then the samples were freeze-dried to obtain anhydrous samples. Both hydrogels before and after ultrasound treatment were observed by SEM. 3.5 Rheology Test of Hydrogels The hydrogels were performed on HAKKE rheometer to study the rheological property. The viscoelastic properties of the hydrogels were measured by performing strain sweep experiments in the oscillation mode. The frequency was set at 1 Hz, and the storage modulus ( G ′) and loss modulus ( G ″) values were recorded by sweeping tests changing the strain from 0.1–1000%. The hydrogels were divided into two groups, one untreated and the other sonicated by ultrasound diagnostic equipment (1.0 mW/cm 2 , 10 min). 3.6 Swelling Behavior Studies of Hydrogels Anhydrous hydrogels after freeze-drying were weighed (W D ), and then stored in PBS buffer to allow water uptake. The swollen hydrogels were extracted and weighed (W S ) after wiping to remove excessive water at different time points, and the W S /W D ratio was calculated. The experiment was measured continuously for 10 h to fully analyze the swelling behavior of the hydrogels. 3.7 Biodegradation Behavior of Hydrogel Anhydrous hydrogels after freeze-drying were weighed (W 0 ) and immersed in PBS buffer (pH = 7.4) with 0.4 mg/mL of lysozyme (1 × 10 5 U·mg) at 37 ℃. The hydrogels were taken out and weighed (W s ) at different times. The experiment was measured continuously for 6 d to fully analyze the biodegradation behavior of the hydrogels. The degradation rates were calculated using the following formula: The degradation rate = (1 – W s /W 0 ) × 100% 3.8 In vitro Drug Release The in vitro drug release behavior of the pH/ultrasound dual-responsive hydrogel were investigated using methylene blue (MB) as the model drug. First, a range of concentrations of MB from 0 to 25 µg/mL were prepared. And the absorbance was measured by UV-visible spectroscopy (INSEA, China). The peak absorbance at 465 nm were recorded to fit the calibration curve of MP. And then, 20 µL of MB solution (1.0 mg/mL) was added into the 980 µL of the uncross-linked hydrogel solution to prepare MB-loaded hydrogel (20 µg MB per 1.0 mL hydrogel). The hydrogel of free releasing was set as control. The MB-loaded hydrogels were immersed in 5.0 mL PBS buffer (pH 7.4) to study release behavior. And hydrogels of ultrasound treatment, pH treatment (immersing into pH 6.0 PBS buffer), ultrasound + pH treatment were set as experimental group. The ultrasound treatment was set at 60th, 120th, 180th, and 300th min, and each ultrasound treatment lasted 10 min. The absorbance of MB in the were measured by UV-visible spectroscopy and the released percentages were calculated by the fitted calibration curve. 3.9 Cell Acquisition and Culture As previously described 30 , 38 , human umbilical cord mesenchymal stem cells (Cyagen, HUXUC-01001, China) were purchased from Cyagen Biotechnology Company. The HUC-MSCs were cultured in MesenCult™ MSC Basal Medium (Stemcell Technologies, RC200133, China). The multilineage differentiation potential of HUC-MSCs was verified by inducing osteogenic, chondrogenic, and adipogenic differentiation using differentiation media. Surface markers of HUC-MSCs (CD105, CD73, CD90, CD45, CD34, HLA-DR) were detected using flow cytometry.C2C12 myoblast cell line was purchased from Procella (Procella, CL-0044, China). The cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/mL streptomycin. When the myoblasts reached 70%-80% confluence, they were induced to differentiate into myotubes using DMEM supplemented with 2% horse serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin. RAW cells (RAW 264.7, a murine-derived macrophage cell line) were obtained from the cell bank of the Chinese Academy of Sciences Typical Culture Preservation Committee. 3.10 EVs Isolation and Characterization The supernatant from HUC-MSCs culture was collected for EVs isolation using ultracentrifugation. The specific steps were as follows: The collected cell supernatant was centrifuged at 300 g, 4°C for 10 minutes to remove cell debris. The supernatant was then centrifuged at 2000 g, 4°C for 10 minutes to remove dead cell debris. Subsequently, the supernatant was centrifuged at 10,000 g, 4°C for 30 minutes to remove cellular debris. The resulting supernatant was filtered through a 0.22 µm filter and transferred to ultracentrifuge tubes. Ultracentrifugation was performed at 100,000 g (Beckman Coulter, USA) for 70 minutes at 4°C. The pellet obtained after centrifugation was resuspended in 1ml PBS 52 . Next, Nanosight NS300 system (Malvern, UK) was used for nanoparticle tracking analysis (NTA) to analyze the size distribution and particle concentration of the vesicles. Western blotting was then performed to detect the surface markers CD63, CD81, and CD9 of the EVs (Abcam, USA). 3.11 PKH-26 Staining and Cellular Uptake Assay The PKH-26 staining kit (Sigma-Aldrich, D0030, China) was employed to label the EVs. The brief procedure, as provided by the manufacturer, is outlined below: The previously isolated EVs were diluted with Dilution C solution, followed by the addition of 4 µL of PKH-26 dye solution. The mixture was then incubated at room temperature in the dark for 5 minutes. Subsequently, the staining reaction was terminated by adding 500 µL of 1% BSA solution. The labeled EVs were pelleted by centrifugation at 1000000 g for 70 minutes at 4°C, followed by resuspension in 200 µL of cold PBS. C2C12 cells were co-cultured with the labeled EVs for 24 hours. The cells were then fixed with 4% paraformaldehyde, stained with DAPI for 5 minutes to label the cell nuclei, and observed under a fluorescence microscope to visualize the stained cells. 3.12 MiRNA Sequencing Analysis Total RNA was extracted from the tissue using TRIzol® Reagent according the manufacturer’s instructions. Then RNA quality was determined by 5300 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). Only high-quality RNA sample was used to construct sequencing library. RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer’s instructions (Illumina, San Diego, CA). 3.13 Proliferation Experiment of C2C12 Cells The proliferation assay of C2C12 cells was conducted using the CCK-8 cell counting kit (CCK-8, Dojindo, Japan) to assess the proliferation of cells stimulated by EVs. In brief, 5000 cells were seeded in a 96-well plate and cultured for 24 hours. Subsequently, 10 µL of CCK-8 solution was added to each well, followed by co-incubation with the cells for 3 hours. Cell proliferation was determined by measuring the absorbance at 450 nm using an enzyme-linked immunosorbent assay plate reader. The data were expressed as mean ± standard deviation (SD) from three independent replicates. 3.14 Immunofluorescence Experiment of C2C12 Cells The immunofluorescence experiment of C2C12 cells (MYOG, DAPI staining) proceeded as follows: After differentiation in high-glucose DMEM medium containing 10% FBS for 3 days, cells in logarithmic growth phase were seeded onto coverslips to form a monolayer. Subsequently, cells were cultured in high-glucose DMEM medium supplemented with 2% horse serum for 7 days. Following this, cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed three times with 1× PBS for 5 minutes each. Non-specific sites were then blocked with 1% bovine serum albumin (BSA) at room temperature for 30 minutes. Each coverslip was incubated overnight at 4°C with mouse monoclonal anti-myogenin primary antibody (1:200 dilution in 1% BSA/PBS) (Beyotime, AF7542, China), followed by three washes with 1× PBS for 5 minutes each. Subsequently, coverslips were incubated with FITC-conjugated secondary antibody (anti-rabbit or anti-mouse) (1:500 dilution in 1% BSA/PBS) (Beyotime, A0562, China) at room temperature in the dark for more than 1 hour, followed by three washes with 1× PBS for 5 minutes each. Finally, coverslips were stained with DAPI (Beyotime, C1005, China) in the dark for 10 minutes, mounted with a mounting medium, and observed and photographed under an immunofluorescence microscope. 3.15 Hydrogel's Protective Effect Against ROS in C2C12 Cells C2C12 cells were seeded in a plate and treated with H 2 O 2 (400 µmol) for 24 hours to establish an oxidative stress microenvironment. Different concentrations of decellularized extracellular matrix materials were then added, and the cells were co-cultured for 48 h. Cell viability was assessed using the CCK-8 and Calcein-AM/PI dual staining method. Intracellular ROS detection was performed using DCFH-DA as the ROS fluorescent probe. Cells were co-incubated with different treatments for 30 minutes, followed by PBS washing. Fluorescence microscopy was used to observe and image the staining of live/dead cells and ROS. Quantitative analysis was conducted using ImageJ software. 3.16 Effect of Hydrogel on Macrophage Polarization The expression of the macrophage marker CD86 (M1) in RAW cells was detected using flow cytometry. RAW cells were seeded in a 6-well plate at a density of 1×10 6 cells/mL with 2 mL per well. After 12 h, the medium was replaced with hydrogel extract, and cells were cultured for an additional 3 days. Subsequently, the cells were scraped off, washed twice with phosphate-buffered saline (PBS), and thoroughly resuspended in 250 µL of fixation/permeabilization solution, followed by incubation at 4°C for 20 minutes. After washing the cells twice with buffer (from the fixation/permeabilization kit, BD), they were incubated with antibody solution containing CD86 (dilution factor 1:100, H2316, Santa Cruz Biotech) at 4°C for 30 minutes. After antibody incubation, the cells were washed twice with buffer, resuspended in PBS, and analyzed using a flow cytometer (FACS, AriaII, BD). 3.17 Animal Model Establishment and Grouping This experiment obtained ethical approval from the Ethics Committee of the Tenth People's Hospital of Shanghai (SHDSYY-2023-3825-3). The specific steps of the experiment are as follows: After sodium pentobarbital anesthesia of SD rats, an incision was made on the left hind limb posterior lateral thigh to expose the sciatic nerve, freeing the sciatic nerve while preserving the branches of the sciatic nerve to the thigh muscles. The proximal end of the nerve was freed to the level of the hamstring tendon, and the distal end was carefully freed to the entry points of the nerve branches into the muscles. The left sciatic nerve was cut off at the distal end below the hamstring tendon, and the proximal end of the nerve was inverted and buried in the nearby hamstring muscle belly. Two nylon sutures were used to secure the outer membrane and fascicles of the distal end of the nerve, which was pulled proximally and fixed to the hamstring tendon, ensuring that the distal end of the sciatic nerve was positioned at the distal end of the hamstring tendon. Before closing the wound, the left hind limb was maximally moved to ensure secure fixation of the traction lines. The experimental groups were as follows: control group, surgery only without any treatment; material group, injection of UR-gel immediately after surgery without ultrasound treatment; UR-gel + ultrasound group, injection of UR-gel immediately after surgery, followed by 15 minutes of 1 MHz ultrasound stimulation to the tibialis anterior muscle at the 2nd week, with the remaining treatment the same as before; EVs@UR-gel + ultrasound group, injection of EVs@UR-gel immediately after surgery, with the remaining treatment the same as before. 3.18 Measurement of Sciatic Nerve Function Index (SFI) A wooden trough with open ends, measuring 60 cm in length, 10 cm in width, and 10 cm in height, was constructed. A piece of white paper weighing 70 g was cut to the same length and width as the trough and laid at the bottom. Rats' hind limbs were colored by dipping them in paint at the ankle joints. The rats were then placed at one end of the trough and allowed to walk towards the other end, leaving 5–6 footprints on each side. The following six parameters were measured for each clear footprint: ETS (injured toe spread), NTS (normal toe spread), EPL (injured print length), NPL (normal print length), EIT (injured intermediary toe spread), NIT (normal intermediary toe spread).These indices were then input into the Bain formula to calculate the SFI. An SFI of 0 indicates normal function, while − 100 indicates complete damage. The Bain formula is as follows: TSF (toe spread factor) = (ETS-NTS)/NTS; PLF (print length factor) = (EPL-NPL)/NPL; ITF (intermediary toe spread factor) = (EIT-NIT)/NIT; SFI = 109.5 TSF − 38.3 PLF + 13.3 ITF − 8.8. 3.19 Measurement of Maximum Isometric Contraction Force A Kocher needle with a diameter of 1.0 mm was used to secure the distal end of the rat's femur and ankle joint to a wooden board, stabilizing the lower leg segment and maintaining muscle length. Simultaneously, the muscle tendon insertion point of the anterior tibial muscle was surgically exposed. The initial stimulation frequency was set at 10 Hz, with a duration of 0.4 milliseconds, and the voltage was set at 2 V. We calibrated the force transducer using weights of 0 g, 10 g, 20 g, 30 g, and 50 g, respectively. The muscle tendon insertion point of the anterior tibial muscle was connected to the force sensor, and a single stimulation of the proximal end of the sciatic nerve anastomosis was applied using the stimulating electrode, maintaining the aforementioned stimulation parameters. We incrementally increased the weight at the muscle tendon insertion point by 0 g, 5 g, 10 g, 15 g, and 20 g increments (increasing by 5 g each time until the optimal length was determined) to find the maximum force generated by this single stimulation at each weight. The muscle length at this point was considered the optimal length. At the optimal length, the voltage was adjusted to 10 V, and the stimulation frequency was varied at 50 Hz, 100 Hz, 150 Hz, and 200 Hz (starting from 50 Hz and increasing by 50 Hz each time) to measure the maximum isometric contraction force under continuous stimulation of the sciatic nerve at equal lengths. 3.20 Muscle Circumference, Muscle Wet Weight and Histological Examination The initial length of the muscles at relaxation was measured. Bilateral anterior tibial muscles were excised completely from their origin to insertion points in relaxed muscles. The superficially adherent subcutaneous tissues were carefully removed, then measure the muscle circumference at the thickest part of the muscle and compare it to the unoperated side, and subsequently, the wet weight was measured by using an analytical balance (with a sensitivity of 1 mg, R200D, Germany) and recorded. Frozen sections from the medial heads of the left anterior tibial and gastrocnemius muscles were subjected to Masson's trichrome staining. Acid fuchsin stained muscle fibers red, while aniline blue stained collagen fibers blue. 3.21 Immunofluorescence Staining of Tissue Sections Tissue sections from the anterior tibial muscle collected at six weeks post-operation were subjected to immunofluorescence staining. The muscle tissue was fixed in 4% formaldehyde, dehydrated through a sucrose gradient, embedded in OCT compound, and sliced into 12-micrometer sections. These sections were then blocked at room temperature for 1 hour. Subsequently, they were incubated overnight at 4°C with primary antibodies, including mouse anti-MyHC (dilution 1:200, Abcam, Cambridge, UK) and rabbit anti-Col-1 (dilution 1:200, Abcam, Cambridge, UK). After rinsing with PBS, the sections were incubated with the corresponding secondary antibodies at room temperature in the dark for 2 hours. Following another round of PBS washing, the sections were stained with DAPI to visualize the cell nuclei. Finally, fluorescence microscopy was employed for imaging. 3.22 Transcriptome Sequencing and Bioinformatics Analysis At 6th week post-operation, total RNA was extracted from denervated and denervated + EVs@UR-gel anterior tibial muscles using mRNA isolation kit. Subsequently, the RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Ribosomal RNA was enzymatically digested using the TruSeq Stranded Total RNA and Ribo-Zero Gold kits. The fragmented RNA was used as a template for cDNA synthesis and library construction with fragment buffer solution. RNA libraries were then subjected to RNA identification using the Agilent 2100 Bioanalyzer. Sequencing was performed using an Illumina sequencer (HiSeqTM 2500 or Illumina HiSeq X Ten). DESeq software was employed for normalizing mRNA counts for each sample and calculating fold changes. Differential expression of reads between the two groups was assessed using a negative binomial distribution test. Finally, genes with fold changes of either > 1.5 or <-1.5, with a q-value < 0.05, were selected as differentially expressed genes. For functional analysis based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, enrichment was considered significant for p-values < 0.05. Conclusion We discovered that the miRNA target genes in HUC-MSCs-EVs are associated with cellular energy metabolism and proliferation pathways, suggesting their potential in preventing denervated muscle atrophy. To efficiently load, protect, and deliver HUC-MSCs-EVs, we developed an ultrasound/pH dual-responsive anti-inflammatory injectable hydrogel. This hydrogel exhibited desirable anti-inflammatory properties and ultrasound controllability. At the early stage of the disease, the hydrogel's pH responsiveness was achieved. The anti-inflammatory and ROS-neutralizing effects of the hydrogel were observed at the initial stage of the disease. At the subsequent stage of muscle atrophy, ultrasound intervention enabled the modifiable release of EVs, which aided in maintaining muscle function and structure after neuromuscular atrophy and enhanced the therapeutic efficacy of HUC-MSCs-sEVs. Overall, the EVs@UR-gel demonstrates significant potential in preventing denervated muscle atrophy and preserving muscle function. Declarations Disclosure of Interest The authors reported no potential conflict of interest. Author Contributions Z.B., J.J., and W.L. contributed equally to this work. Z.B., J.J., W.L., F.Z., M.S., J.D., and P.W. designed the research; Z.B., J.J., W.L., J.H., Z.Z., J.H., J.A., J.H., and J.Y. performed the research; F.Z., M.S., J.D., and P.W. analyzed the data; Z.B., J.J., W.L., F.Z., M.S., J.D., and P.W. wrote the paper. Acknowledgements This research was supported by National Natural Science Foundation of China (52222306, 22075212, 21925505 and 22305177), the international scientific collaboration fund of Science and Technology Commission of Shanghai Municipality (21520710100, 23520710900), the fellowship of China Postdoctoral Science Foundation (2022M720107, GZB20230517) and Shanghai“Super Postdoc” Incentive Plan (2022568), Shanghai Rising-Star Program (Sailing, 23YF1433000), Natural Science Foundation of Shanghai (20ZR1443200). J.D. is the recipient of National Science Fund for Distinguished Young Scholars. References Martin JA et al (2018) Gauging force by tapping tendons. Nat Commun 9:1592 Cisterna BA et al (2020) Active acetylcholine receptors prevent the atrophy of skeletal muscles and favor reinnervation. 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Chem Rev 123:834–873 Lobb RJ et al (2015) Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles 4:27031 Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files image1.png Scheme 1 | The illustration of bioactive EVs loading dual-responsive hydrogel used in the staged treatment of denervated muscle atrophy. a The preparation process of pH/ultrasound dual-responsive and HUC-MSCs-EVs loaded injectable hydrogel and its in vivo administration method. bThe programmed treatment of HUC-MSCs-EVs in denervated muscle atrophy model with sonication. In the initial phase of denervated muscle atrophy, the hydrogel scavenged ROS generated by hypoxia. Subsequently, during the second phase of inflammatory response, the hydrogel facilitated inflammatory remodeling. In the third and fourth phases, subsequent to sonication, a considerable number of EVs were released, which facilitated the remodeling of the adult muscle and extracellular matrix to maintain muscle function and prevent irreversible myasthenia. c The therapeutic efficacy of the material was analyzed in terms of the functional and anatomical structure of the skeletal muscle by rat footprint analysis, maximum muscle tone measurements, and immunofluorescence staining of the specimens. SeparateSourceDatafiles.rar Dataset 1 supportingfigures.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4715142","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":333945153,"identity":"69ac21f9-d7a3-4a82-aca9-97a3f4aa9484","order_by":0,"name":"Jianzhong 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1","display":"","copyAsset":false,"role":"figure","size":368800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterizations and ultrasound-responsiveness study of UR-gel.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e FTIR spectra of OCS, CS, CMCS, and UR-gel. \u003cstrong\u003eb-c\u003c/strong\u003e The SEM images of UR-gel \u003cstrong\u003e(b) \u003c/strong\u003ebefore ultrasound stimulation and \u003cstrong\u003e(c) \u003c/strong\u003eafter ultrasound stimulation. \u003cstrong\u003ed\u003c/strong\u003e Rheological property of hydrogels with different treatments. \u003cstrong\u003ee\u003c/strong\u003e Swelling ratio of UR-gel immersed in PBS buffer (pH 7.4). \u003cstrong\u003ef\u003c/strong\u003eDegradation rate of UR-gel in PBS buffer containing lysozyme. \u003cstrong\u003eg\u003c/strong\u003e The demonstration of injectable properties. \u003cstrong\u003eh\u003c/strong\u003e Optical images of the UR-gel and Dox-loaded UR-gel. \u003cstrong\u003ei\u003c/strong\u003e Optical images of the UR-gel and Dox-loaded UR-gel under ultraviolet light. \u003cstrong\u003ej\u003c/strong\u003e The drug release behavior of UR-gel under different conditions, and the accelerated release was observed at each ultrasound-stimulation time point.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4715142/v1/f56c56759de6973c4e93cbe3.jpeg"},{"id":61559201,"identity":"3c40ddce-7e39-4396-a868-b69b6c919349","added_by":"auto","created_at":"2024-08-01 08:16:56","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":596842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEVs extracted from HUC-MSC has high biological activity to promote the migration, proliferation and differentiation of C2C12 cells. a-b (a)\u003c/strong\u003eTEM image and\u003cstrong\u003e (b)\u003c/strong\u003e NTA result of EVs, scale bar = 100 nm. \u003cstrong\u003ec\u003c/strong\u003e The typical protein (CD81, CD63, CD9) markers analysis of EVs with HUC-MSC cells as control. \u003cstrong\u003ed\u003c/strong\u003e Sequencing results of miRNA within EVs. \u003cstrong\u003ee\u003c/strong\u003e the phagocytosis of EVs by C2C12 cells, scale bar = 25μm. \u003cstrong\u003ef\u003c/strong\u003eTranswell experiment of EVs on C2C12 cell migration，scale bar=150μm. \u003cstrong\u003eg\u003c/strong\u003eAnalysis of the quantity of C2C12 cell migration influenced by EVs. \u003cstrong\u003eh\u003c/strong\u003eExperiment showing the effect of EVs on cell proliferation，scale bar=100μm. \u003cstrong\u003ei\u003c/strong\u003e Cell proliferation assay using CCK8 influenced by EVs. \u003cstrong\u003ej\u003c/strong\u003e Experiment demonstrating the promotion of C2C12 cell differentiation by EVs，scale bar=100μm (blue represents DAPI staining, green represents MYOG staining). **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 vs. the control. Data are presented as the mean ± standard.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4715142/v1/50e545af7b8b14aac6865a08.jpeg"},{"id":61558677,"identity":"e15b7416-501a-4e03-b15e-065ba9ebf220","added_by":"auto","created_at":"2024-08-01 08:08:56","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":698643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEVs@UR-gel showed high biological activity in vitro. a \u003c/strong\u003eGrowth status of C2C12 cells on the scaffold after seeded for3 days. Phalloidin visually describes the cytoskeleton, DAPI evaluates the number of cells, and 3D displays the depth of cell growth through confocal microscope z-axis photography, scale bar=100μm.\u003cstrong\u003e b \u003c/strong\u003eActivity of cells co-cultured with the scaffold. \u003cstrong\u003ec\u003c/strong\u003e Number of cells co-cultured with the scaffold. \u003cstrong\u003ed\u003c/strong\u003e Depth of cell growth on the scaffold.\u003cstrong\u003e e \u003c/strong\u003eTranswell experiment of C2C12 cells with different materials. \u003cstrong\u003ef\u003c/strong\u003e Quantity of C2C12 cell migration. \u003cstrong\u003eg \u003c/strong\u003eDifferentiation effect of C2C12 cells with different materials. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. the UR-gel, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 vs. the UR-gel. Data are presented as the mean ± standard.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4715142/v1/30d6a3107f0b04140f51aaef.jpeg"},{"id":61558683,"identity":"9b0e3b50-cad7-4094-8f05-f7fc096b43b4","added_by":"auto","created_at":"2024-08-01 08:08:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1555046,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUR-gel has anti-inflammatory and antioxidant abilities. a\u003c/strong\u003e Live/dead staining of C2C12 cells, the blank group was set as control, the green fluorescence represented living cells, and the red fluorescence represented dead cells, indicating that UR-gel had a protective effect on C2C12 cell death induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, scale bar = 100 μm.\u003cstrong\u003e b \u003c/strong\u003eCell viability assay after co-cultured with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 h. \u003cstrong\u003ec\u003c/strong\u003e Intracellular ROS scavenging ability of UR-gel in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced C2C12 using DCFH-DA as a fluorescent ROS probe, scale bar = 100 μm. \u003cstrong\u003ed\u003c/strong\u003e Intensity of ROS fluorescence. \u003cstrong\u003ee\u003c/strong\u003e After being incubated with 100 ng mL\u003csup\u003e−1\u003c/sup\u003e LPS for 24 h beforehand, RAW 264.7 cells treated with PBS, UR-gel and UR-gel+US, and analyzed by immunofluorescent staining of phalloidin (red) and DAPI (blue), scale bar = 100 µm. \u003cstrong\u003ef\u003c/strong\u003e flow cytometry of CD86 expression. \u003cstrong\u003eg\u003c/strong\u003e Schematic diagram of the gel immune regulation process.\u003cem\u003e **p\u003c/em\u003e\u0026lt;0.01 vs. the control. Data are presented as the mean ± standard.\u003c/p\u003e","description":"","filename":"image5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4715142/v1/43b2add82a7f3ae2ea2acd2d.jpg"},{"id":61558679,"identity":"9da42475-9aba-4981-9680-fd2fe47e4daa","added_by":"auto","created_at":"2024-08-01 08:08:56","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":295818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEVs@UR-gel promotes the recovery of motor function. a\u003c/strong\u003e Photograph of tibialis anterior muscle from rats after treatment with various strategies for 6 weeks and 12 weeks in different groups. \u003cstrong\u003eb\u003c/strong\u003e Proportion of maximal muscle tension retained in the tibialis anterior muscle of rats.\u003cstrong\u003e c\u003c/strong\u003eDiameter of the tibialis anterior muscle in rats. \u003cstrong\u003ed\u003c/strong\u003e Wet weight of the tibialis anterior muscle in rats. \u003cstrong\u003ee\u003c/strong\u003e SFI analysis of rats at 12 weeks. \u003cstrong\u003ef-g\u003c/strong\u003e Numerical analysis related to SFI in rats. \u003cem\u003e*p\u003c/em\u003e\u0026lt;0.05 vs. the control; \u003cem\u003e**p\u003c/em\u003e\u0026lt;0.01 vs. the control. Data are presented as the mean ± standard.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4715142/v1/ef1664e36e369feecba3a931.jpeg"},{"id":61558678,"identity":"686cda00-7cd6-41ac-be48-c87c6f8f4164","added_by":"auto","created_at":"2024-08-01 08:08:56","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":729143,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of EVs@UR-gel on muscle regeneration and type I collagen deposition in target muscle in vivo. a\u003c/strong\u003e after treatment for 6 weeks, the tissue sections stained with Masson's trichrome and immunofluorescence (green: MyHC immunofluorescence staining; red: Col-1 immunofluorescence staining; blue: DAPI staining; merged image), EVs@UR-gel can promote the regeneration of MyHC and inhibit the deposition of COL-1\u003cstrong\u003e, \u003c/strong\u003escale bar = 100μm. \u003cstrong\u003eb-c \u003c/strong\u003eThe semi-quantification of \u003cstrong\u003e(b)\u003c/strong\u003e MyHC and \u003cstrong\u003e(c) \u003c/strong\u003eCol-1 immunofluorescence signal intensity. \u003cstrong\u003ed-e\u003c/strong\u003e Go analysis of changes in transcriptome profile of target muscle treated by\u003cstrong\u003e \u003c/strong\u003eEVs@UR-gel+US. group and the control group -in \u003cstrong\u003e(d) \u003c/strong\u003eup- and \u003cstrong\u003e(e) \u003c/strong\u003edown- regulated genes. \u003cstrong\u003ef\u003c/strong\u003e Differentially expressed genes (DEGs) analysis between the EVs@UR-gel+US. group and the control group. \u003cstrong\u003eg\u003c/strong\u003e GO Chord plot of interested GO terms and the corresponding DEGs.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4715142/v1/2074b922bbfecf96fd0fd198.jpeg"},{"id":63495875,"identity":"eb812842-898c-489a-a28e-6596d4daa7ad","added_by":"auto","created_at":"2024-08-28 20:03:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5184772,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4715142/v1/db70f548-9193-4709-be42-b8bf09bc659b.pdf"},{"id":61558682,"identity":"4d669b87-fd7f-454a-862c-b9a0b5069056","added_by":"auto","created_at":"2024-08-01 08:08:56","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2871182,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1 |\u003c/strong\u003e \u003cstrong\u003eThe illustration of bioactive EVs loading dual-responsive hydrogel used in the staged treatment of denervated muscle atrophy.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e The preparation process of pH/ultrasound dual-responsive and HUC-MSCs-EVs loaded injectable hydrogel and its \u003cem\u003ein vivo \u003c/em\u003eadministration method. \u003cstrong\u003eb\u003c/strong\u003eThe programmed treatment of HUC-MSCs-EVs in denervated muscle atrophy model with sonication. In the initial phase of denervated muscle atrophy, the hydrogel scavenged ROS generated by hypoxia. Subsequently, during the second phase of inflammatory response, the hydrogel facilitated inflammatory remodeling. In the third and fourth phases, subsequent to sonication, a considerable number of EVs were released, which facilitated the remodeling of the adult muscle and extracellular matrix to maintain muscle function and prevent irreversible myasthenia. \u003cstrong\u003ec \u003c/strong\u003eThe therapeutic efficacy of the material was analyzed in terms of the functional and anatomical structure of the skeletal muscle by rat footprint analysis, maximum muscle tone measurements, and immunofluorescence staining of the specimens.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4715142/v1/66aa620272595ee7bf5abf67.png"},{"id":61558681,"identity":"5bad041d-364b-422b-b7e0-363c0db1aed3","added_by":"auto","created_at":"2024-08-01 08:08:56","extension":"rar","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2443167,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 1\u003c/p\u003e","description":"","filename":"SeparateSourceDatafiles.rar","url":"https://assets-eu.researchsquare.com/files/rs-4715142/v1/d199c724440f3ef5e7a2ac21.rar"},{"id":61558684,"identity":"8a84bdbf-f4af-4b52-8e22-405adae39d61","added_by":"auto","created_at":"2024-08-01 08:08:56","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":7991435,"visible":true,"origin":"","legend":"","description":"","filename":"supportingfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-4715142/v1/0123d762bc7a75c1f0357673.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Treatment of Denervated Muscle Atrophy by Injectable Dual-responsive Hydrogels Loaded with Extracellular Vesicles","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSkeletal muscles are essential for human motion, executing various postures and generating and ceasing actions\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Muscle atrophy from nerve damage is prevalent and linked to disrupted nerve supply, contributing significantly to global disability and economic burden\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Early nerve repair is beneficial, but factors like immature surgical conditions and slow nerve regeneration can lead to muscle atrophy\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Current treatments focus on neural repair but lack strategies for target muscle\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Conventional clinical methods such as passive skeletal muscle exercises and electrical stimulation of atrophied muscles fail to meet the needs of both patients and clinical practitioners\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Denervated muscles will undergo a staged, highly programmed and complex pathological process, which may lead to irreversible atrophy and lifelong motor dysfunction if it exceeds the self-healing ability\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, current treatments simply target specific stages of the disease, lacking a comprehensive approach to inhibit denervated muscle atrophy from multiple perspectives.\u003c/p\u003e \u003cp\u003eMesenchymal stem cell (MSC) therapy has shown potential in treating muscle atrophy, but it faces challenges related to cell source, tissue origin, and cellular heterogeneity\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. EVs derived from stem cells offer comparable benefits without the issues associated with live cell use\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Moreover, EVs contain miRNAs, ncRNAs and growth factors, playing an important role in cell communication and regulating cell function\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Initial research indicates that these contents are enriched in pathways related to cellular metabolism and may counteract atrophic changes, with their reparative effects being dose-dependent\u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34 CR35 CR36\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Our preliminary research also indicates that treating bone and cartilage injuries with varying concentrations of EVs results in better treatment outcomes with higher concentrations significantly outperforming lower concentrations\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. However, the in vivo stability and bioavailability of EVs are compromised, necessitating encapsulation in hydrogels for tunable release and sustained bioactivity\u003csup\u003e\u003cspan additionalcitationids=\"CR40 CR41 CR42\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Therefore, the development of EVs capable of early anti-inflammatory and antioxidant action, along with sustained \"burst release\" of EVs at specific stages for the purpose of programmed phased therapeutic strategies, is essential for the clinical application of EVs therapy.\u003c/p\u003e \u003cp\u003eBioengineering has shown promise in treating musculoskeletal injuries, but tailored material requirements for muscle repair, which demands high compliance and fatigue resistance, are still under investigation\u003csup\u003e\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The initial research findings indicate that injectable hydrogels can respond to repeated mechanical stress through a process known as mechanochemical conversion. This enables external intervention to induce the transition of injectable hydrogels from a sol state to a gel state, which in turn facilitates the filling, curing, and drug release processes\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In the context of denervated muscle atrophy, the distinctive pathophysiology and evident staging of the condition necessitate the development of biomaterials that are injectable, possess anti-inflammatory and antioxidative properties, and are capable of dynamically and precisely regulating EVs release based on real-time therapeutic efficacy assessment during specific time intervals.\u003c/p\u003e \u003cp\u003eIn light of this concept and the pressing clinical demands, we demonstrated that umbilical cord mesenchymal stem cells-derived EVs (HUC-MSCs-EVs) effectively promote the migration, proliferation, and differentiation of myoblasts. Then we prepared a pH/ultrasound dual-responsive hydrogel (UR-gel) as the EVs carrier, achieving early anti-inflammatory and antioxidative effects at the lesion site through pH response. Additionally, based on the therapeutic efficacy, we utilized ultrasound at specific stages to achieve instantaneous burst release of HUC-MSCs-EVs, combating muscle atrophy and maintaining muscle function and morphology. This demonstrates the effectiveness of treating denervated muscle atrophy by addressing the disease's pathophysiological processes and utilizing bioactive materials. It offers new avenues and methods for clinical denervated muscle atrophy treatment. This comprehensive therapeutic approach combines the characteristics of biomaterials with the bioactivity of HUC-MSCs-EVs, with the potential to improve the quality of life for patients suffering from denervated muscle atrophy and to slow disease progression. Further research and clinical practice may lead to this treatment method becoming a significant therapeutic intervention for denervated muscle atrophy.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Synthesis and Characterizations of UR-gel\u003c/h2\u003e \u003cp\u003eThe synthesis of UR-gel was illustrated in scheme 1. Oxidized chondroitin sulfate (OCS) was first obtained from chondroitin sulfate (CS) by NaIO\u003csub\u003e4\u003c/sub\u003e oxidation. As shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the stretching vibration band of aldehyde group at 1736.5 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e appeared in the FTIR spectrum of the OCS, demonstrating successful oxidation. Then, the hydrogel was synthesized by mixing OCS, carboxymethyl chitosan (CMCS) and cystamine dihydrochloride solutions. The hydrogel was spontaneously crosslinked in situ through the Schiff base reaction between the amino and aldehyde groups. The FTIR spectrum of the UR-gel showed the disappearance of the characteristic peak at 1736.5 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which demonstrated that the aldehyde groups of OCS were fully reacted by the amino groups of CMCS as well as cystamine dihydrochloride, and the successful crosslinking of UR-gel.\u003c/p\u003e \u003cp\u003eObvious porous network was observed in SEM image of hydrogel as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, which could be used to encapsulate EVs within the cavity. In addition, we observed the morphology of the UR-gel after ultrasound stimulation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, the pore sizes of the UR-gel increased after ultrasound stimulation. This is because the ultrasound stimulation partially destroyed the cross-linking structure of the UR-gel\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Meanwhile, the rheological properties of the hydrogels were measured before and after ultrasound stimulation. As shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, the storage modulus (\u003cem\u003eG\u003c/em\u003e\u0026prime;) is greater than loss modulus (\u003cem\u003eG\u003c/em\u003e\u0026prime;\u0026prime;) at a strain of 0.1%. With increasing strain to 1002.14%, \u003cem\u003eG\u003c/em\u003e\u0026prime; is smaller than \u003cem\u003eG\u003c/em\u003e\u0026prime;\u0026prime;, suggesting that UR-gel structure was destroyed. Furthermore, \u003cem\u003eG\u003c/em\u003e\u0026prime; of hydrogel after ultrasound stimulation was lower than that before ultrasound stimulation, indicating that ultrasound could reduce its storage modulus and accelerate the destroying of hydrogels.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, swelling study revealed that the UR-gel could absorb large amount of water, and the water absorption rate maintained at about 50 times after 8 h and onwards, which indicated that UR-gel had great swelling performance and drug storage capacity. We also investigated the biodegradability with UR-gel immersing in PBS buffer containing 0.4 mg/mL lysozyme. The degradation rate increased with increasing time and reached 69.92% after immersing 6 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Furthermore, since the UR-gel was cross-linked by reversible covalent bonds, the UR-gel had the ability to realize the injection capacity as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition, we investigated the drug release behavior of UR-gel using doxorubicin as the model drug (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). The fluorescence under an ultraviolet light (365 nm) was observed, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei. It showed that model drug could load into UR-gel. The drug release rate was investigated using methylene blue as model drug. The rate was calculated based on the standardized fitting curve of a range of concentrations of methylene blue solutions (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea-b). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej, compared to the release percentage of free releasing, the drug release percentage from the hydrogel increased under ultrasound stimulation or acidic environment (pH 6.0) conditions. Moreover, the ultrasound stimulation and acidic conditions could synergistically promote the drug release percentage, which reach 92.35% release percentage after 420 min incubation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Characteristics of HUC-MSCs-EVs\u003c/h2\u003e \u003cp\u003eHUC-MSCs and HUC-MSCs-EVs were isolated and identified. Under light microscopy, typical spindle-shaped morphology of HUC-MSCs was observed (Fig. S2a). The differentiation potential of HUC-MSCs into cartilage, bone, and fat was evaluated using Alcian blue, Alizarin Red, and Oil Red O staining, respectively (Fig. S2b). Furthermore, flow cytometry was employed to detect surface markers CD105, CD73, CD90, CD45, CD34, and HLA-DR on HUC-MSCs (Fig. S2c). The results demonstrated compliance with the identification criteria for stem cells. Subsequently, EVs were successfully isolated and extracted, and typical cup-shaped structures of EVs were observed under TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). NTA results showed that the diameters of vesicles are mainly in the range of 80\u0026ndash;200 nm, with an average diameter of 142.5 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). WB analysis revealed high expression of EVs surface markers CD63, CD81, and CD9, while they were absent on cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Overall, these results confirm successful isolation of EVs from HUC-MSCs.\u003c/p\u003e \u003cp\u003eSubsequently, sequencing and analysis of miRNA expression levels in HUC-MSCs-EVs were performed and the results revealed that abundant miRNAs are contained in HUC-MSC-EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). It is well known that miRNAs regulate target genes by binding to the 3\u0026rsquo;UTR of target genes or specific sequences of target genes. Multiple databases were used to predict the target genes of miRNAs enriched in HUC-MSCs-EVs (Fig. S3a). GO analysis and KEGG analysis on these target genes showed that target genes of miRNAs abundant in HUC-MSCs-EVs were enriched in pathways related to cell cycle, DNA replication, insulin resistance, and various signaling pathways, indicating their roles in cell migration, proliferation, and differentiation of C2C12 cells (Fig. S3b-c).\u003c/p\u003e \u003cp\u003eFurthermore, the effects of HUC-MSCs-EVs on the migration, proliferation, and differentiation of C2C12 cells were validated. Initially, HUC-MSCs-EVs were labeled by using the PKH-26 staining kit and co-cultured with C2C12 cells for 24 h. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, red fluorescent-labeled EVs were internalized by C2C12 cells, with the cell nuclei stained blue using DAPI and the cell cytoskeleton stained green using FITC-labeled phalloidin. It was observed that the HUC-MSCs-EVs were successfully endocytosed by C2C12 cells, primarily accumulating around the cell nuclei. Transwell migration assays demonstrated a significant enhancement in the migratory capacity of C2C12 cells upon addition of HUC-MSCs-EVs. Moreover, the control groups with varying concentrations of EVs were established to determine the impact on cells. These were designated as EVs1 (5\u0026times;10\u003csup\u003e8\u003c/sup\u003e particles/mL) and EVs2 (1\u0026times;10\u003csup\u003e9\u003c/sup\u003e particles/mL). The results showed that EVs2 significantly enhanced cell migration compared to EVs1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-g), indicating that the concentration is an important factor influencing the therapeutic efficacy of HUC-MSCs-EVs. Edu staining, which reflects cell proliferation and activity, revealed a substantial increase in the number of proliferating cells in both EVs1 and EVs2 groups compared to the control. EVs2 demonstrated a notably higher proliferation rate than EVs1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh), confirming the ability of HUC-MSCs-EVs to promote C2C12 cell proliferation, with higher concentrations showing superior effects. The CCK8 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei) corroborated the previous findings, further validating the conclusion. MYOG, a key marker in myogenic differentiation, showed that the addition of extracellular vesicles positively affected the differentiation of C2C12 cells. There was a positive correlation between vesicle concentration and the extent of myogenic differentiation, as evidenced by immunofluorescence results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). This correlation underscores the sequencing and prediction of HUC-MSCs-EVs, highlighting their potential therapeutic impact on denervated muscle atrophy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.3\u003c/b\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eBiocompatibility of EVs@UR-gel\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo validate the in vitro biocompatibility of EVs@UR-gel, a concentration of 1\u0026times;10\u003csup\u003e10\u003c/sup\u003e particles/ml hydrogel was prepared by adding 10 \u0026micro;L of 1\u0026times;10\u003csup\u003e11\u003c/sup\u003e particles/mL into 90 \u0026micro;L of UR-gel. The UR-gel alone acted as the control, while UR-gel stimulated with ultrasound at an intensity of 1 MHz for 10 minutes and EVs@UR-gel the same stimulated served as experimental groups. After co-culturing with C2C12 cells for 3 days, Actin/DAPI staining results showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) that the cells were well spread on the hydrogel scaffold. Following ultrasound stimulation, there was a noticeable increase in cell number and growth depth, which was further enhanced with the addition of EVs. CCK-8 results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and quantitative analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d) demonstrated an enhanced proliferative capacity of the material post-ultrasound treatment, which was further augmented with the addition of EVs. Quantitatively, compared to the gel group and ultrasound-stimulated gel group, the ultrasound-stimulated EVs@UR-gel group showed an increase in nuclear count from 287\u0026thinsp;\u0026plusmn;\u0026thinsp;36 cells/field to 891\u0026thinsp;\u0026plusmn;\u0026thinsp;31 cells/field and an increase in cell growth depth into the hydrogel from 90.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10 nm to 169.3\u0026thinsp;\u0026plusmn;\u0026thinsp;12.7 nm.\u003c/p\u003e \u003cp\u003eSubsequently, the effects of EVs@UR-gel on C2C12 cell migration and differentiation were verified. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee illustrates that cell migration was promoted by UR-gel after ultrasound treatment, with an even more pronounced effect observed with EVs@UR-gel. Quantitative analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef) confirmed that the EVs@UR-gel group had a significantly stronger effect on cell migration than both the UR-gel and ultrasound-treated UR-gel groups. Differentiation experiments, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg and Fig. S4, demonstrated that MyoG immunofluorescence staining indicated a marked ability of EVs@UR-gel with ultrasound treatment to enhance C2C12 cell differentiation. These results suggest that ultrasound stimulation induces structural changes in UR-gel, increasing its porosity and providing more space for C2C12 cell growth. The release of encapsulated EVs post-ultrasound stimulation aids in cell growth into deeper regions of the hydrogel. It is highlighted that the difference between the UR-gel and EVs@UR-gel groups underscores the protective role of the hydrogel on EVs, indicating that even after 10 minutes of 1 MHz ultrasound stimulation, the biological activity of EVs is preserved.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Anti-inflammatory and Antioxidant Effects of EVs@UR-gel\u003c/h2\u003e \u003cp\u003eThe efficacy of EVs@UR-gel in protecting target muscles from oxidative stress and inflammation following denervation has been validated. At the early stages of denervated muscle atrophy, muscles experience a loss of voluntary contraction and fibrillation abilities, leading to blood accumulation and a hypoxic microenvironment. This hypoxia, over time, triggers inflammatory cell aggregation and a vicious cycle that accelerates muscle atrophy. The capacity of carrier materials to neutralize free radicals is therefore essential. The anti-inflammatory and antioxidant properties of EVs@UR-gel were tested through protective, ROS scavenging, and macrophage polarization experiments on C2C12 cells using UR-gel.\u003c/p\u003e \u003cp\u003eThe anti-inflammatory and antioxidant properties of EVs@UR-gel were tested through protective, ROS scavenging, and macrophage polarization experiments on C2C12 cells using UR-gel. Calcein-AM/PI staining post-H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment showed an increase in live cells with UR-gel addition, and a slight increase post-ultrasound treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Fig. S5a). CCK-8 results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) indicated higher cell viability with UR-gel, regardless of ultrasound treatment, suggesting a protective effect against ROS without significant impact from ultrasound on ROS resistance. Furthermore, 2',7'-dichlorofluorescin diacetate (DCFH-DA) was used to measure intracellular ROS levels, revealing that UR-gel reduced fluorescence intensity, indicating a downregulation of ROS induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-d). The ability of cells to resist ROS is enhanced after ultrasound treatment, likely due to structural changes in the hydrogel after ultrasound, leading to internalization of some hydrogel components by cells and thus enhancing intracellular anti-ROS capacity.\u003c/p\u003e \u003cp\u003eConsidering the association between inflammatory response and M1 macrophage polarization, the effect of materials on anti-inflammatory activity against M1 macrophages was investigated by co-incubating with RAW264.7 cells stimulated with LPS. A significant reduction in pseudopod formation indicated a decrease in the amount of cells polarized to M1 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee and Fig. S5b). Combined with the above results, the application of ultrasound significantly reduced intracellular reactive oxygen species content, restored the characteristics of M0 macrophages, and induced a rounded or ellipsoidal morphology with strong adhesion to the wall, as well as elongated spindle shape, a characteristic morphology of M2 polarization. After fluorescence labeling with anti-CD86, flow cytometry results showed that the proportions of M1 macrophages were 81.2%, 63.3%, and 51.4% in the different treatment groups, respectively, confirming the hydrogel's antioxidant and anti-inflammatory effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). The material's strong anti-inflammatory and antioxidant capabilities are suitable for early treatment strategies of denervated muscle atrophy. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg illustrates the immunomodulation mechanism of UR-gel.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5 The Therapeutic Effects of EVs@UR-gel on Motor Function Recovery in Rats\u003c/h2\u003e \u003cp\u003eTo assess the reparative effect of EVs@UR-gel on denervated muscle atrophy, during model establishment, the hydrogel was injected into the tibialis anterior muscle of rats. Gross specimens showed that the tibialis anterior muscle in the EVs@UR-gel group was significantly thicker compared to other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). After 6 weeks of denervation, measurements indicated that the EVs@UR-gel group had a higher residual muscle circumference, retained passive muscle force, and greater muscle wet weight compared to other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-d). At 6 weeks post nerve repair (12th week), the maximum muscle strength which is closely related to muscle function, the muscle circumference and the wet weight, can be restored to 89.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96%, 76.02\u0026thinsp;\u0026plusmn;\u0026thinsp;7.49% and 88.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65% of the healthy state, respectively. The intervention with EVs@UR-gel reduced the rate of muscle atrophy and loss of muscle strength post-denervation while preserving muscle recovery capability.\u003c/p\u003e \u003cp\u003eTo explore the protective effect of EVs@UR-gel on the motor ability of rats after denervation and repair, gait analysis was conducted on rats at the 12th week. Motor function was assessed using the Sciatic Functional Index (SFI), with 0 indicating normal function and \u0026minus;\u0026thinsp;100 indicating complete loss of motor function. In the assessment of normal motor function, values of PLF, TSF, and ITF were 0. Results indicated that at the 12th week, except for the sham surgery group, all indicators and the comprehensive index SFI in the EVs@UR-gel group were significantly higher than in the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-i). These findings suggest that the designed EVs@UR-gel hydrogel can maintain the motor ability of target muscles after denervation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunofluorescence Staining Results and Gross Specimens\u003c/h2\u003e \u003cp\u003eTissue sections of the tibialis anterior muscle at the 6th week were subjected to Masson's staining and immunofluorescence staining to validate the anatomical protection and maintenance of denervated muscles by EVs@UR-gel. Masson's staining indicated that muscle fibers in the EVs@UR-gel group preserved their integrity, had a tight arrangement, and featured reduced collagen fiber thickness and an organized extracellular matrix, contrasting with the other three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The extent of muscle atrophy was notably reduced, and fibrosis was less pronounced in the EVs@UR-gel group. Further immunofluorescence staining of the sections with MyHC and Col-1 antibodies showed that MyHC fluorescence intensity and morphology were significantly better in the EVs@UR-gel group compared to the other groups. The fluorescence intensity of MyHC increased sequentially from the control group to the UR-gel group, ultrasound-treated UR-gel group, and EVs@UR-gel group, while Col-1 fluorescence decreased. Subsequently, the result was subjected to further clarification through the implementation of quantitative calculations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-c). Tissue-level observations suggest that the early anti-inflammatory effects of EVs@UR-gel are advantageous in mitigating muscle atrophy and fibrosis, with ultrasound-stimulated UR-gel showing enhanced reparative effects. The release of EVs \u003cem\u003ein vivo\u003c/em\u003e, as indicated by the EVs@UR-gel group results, promotes the preservation of target muscle function and morphology. Changes in MyHC, the principal contractile protein, and Col-1, a key component in muscle fibrosis, underscore the protective and maintenance effects of EVs@UR-gel on the function of denervated skeletal muscle. The trend of these changes also supports the correctness of our material design theory.\u003c/p\u003e \u003cp\u003eSubsequently, mRNA sequencing was performed on the control group and EVs@UR-gel group, revealing 560 upregulated genes and 315 downregulated genes (Fig. S6). GO and KEGG analysis of these differentially expressed genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-g) showed significant enrichment in signaling pathways related to muscle tissue development, muscle contraction, anti-inflammatory responses, and metabolic promotion. These findings imply that the application of EVs@UR-gel is capable of sustaining the function of denervated target muscles and stabilizing the internal environment at a genetic level.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Synthesis of Oxidized Chondroitin Sulfate (OCS)\u003c/h2\u003e \u003cp\u003eOCS was synthesized by an oxidation reaction using NaIO\u003csub\u003e4\u003c/sub\u003e. First, 1.0 g of chondroitin sulfate (CS) was dissolved in 20 mL distilled water. Then, 700 mg of NaIO\u003csub\u003e4\u003c/sub\u003e was dissolved in 20 mL distilled water. After dissolving completely, two solutions were mixed and stirred for 6 h in the dark at room temperature. Subsequently, 1 mL of ethylene glycol was added into the reaction system to terminate the oxidation reaction for 1 h. And then, the solution was dialyzed with distilled water for 3 days and freeze-dried, resulting in a white solid sample. OCS was stored at room temperature and protected away from light.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Fabrication of pH/Ultrasound Dual-Responsive Hydrogel\u003c/h2\u003e \u003cp\u003eThe pH/ultrasound dual-responsive hydrogel was prepared based on the principle of the Schiff base reaction. In brief, carboxymethyl chitosan (CMCS, 1.0 g) was dissolved in 50 mL of deionized water with stirring to completely dissolved. OCS (500 mg) was dissolved in 5 mL of deionized water to obtain 100 mg/mL of OCS. Cystamine dihydrochloride was dissolved in deionized water to 1.0 mol/L. And then, CMCS, OCS and cystamine dihydrochloride solutions were mixed at a volume ratio of 8:1:1 at room temperature with slight stirring. The pH/ultrasound dual-responsive hydrogel was gradually solidified and fabricated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Fourier Transform Infrared Spectra (FTIR) Measurement\u003c/h2\u003e \u003cp\u003eFTIR data were recorded using a Bruker Equinox 55 spectrometer at frequencies ranging from 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and resolution of 0.5 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Samples were powdered and mixed with dried KBr powder and pressed into pellet form.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Morphology of Hydrogels\u003c/h2\u003e \u003cp\u003eThe morphologies of hydrogels were analyzed by scanning electron microscopy (SEM, Zeiss microscope). Samples were washed for three time with deionized water and frozen in -20 ℃. Then the samples were freeze-dried to obtain anhydrous samples. Both hydrogels before and after ultrasound treatment were observed by SEM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Rheology Test of Hydrogels\u003c/h2\u003e \u003cp\u003eThe hydrogels were performed on HAKKE rheometer to study the rheological property. The viscoelastic properties of the hydrogels were measured by performing strain sweep experiments in the oscillation mode. The frequency was set at 1 Hz, and the storage modulus (\u003cem\u003eG\u003c/em\u003e\u0026prime;) and loss modulus (\u003cem\u003eG\u003c/em\u003e\u0026Prime;) values were recorded by sweeping tests changing the strain from 0.1\u0026ndash;1000%. The hydrogels were divided into two groups, one untreated and the other sonicated by ultrasound diagnostic equipment (1.0 mW/cm\u003csup\u003e2\u003c/sup\u003e, 10 min).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Swelling Behavior Studies of Hydrogels\u003c/h2\u003e \u003cp\u003eAnhydrous hydrogels after freeze-drying were weighed (W\u003csub\u003eD\u003c/sub\u003e), and then stored in PBS buffer to allow water uptake. The swollen hydrogels were extracted and weighed (W\u003csub\u003eS\u003c/sub\u003e) after wiping to remove excessive water at different time points, and the W\u003csub\u003eS\u003c/sub\u003e/W\u003csub\u003eD\u003c/sub\u003e ratio was calculated. The experiment was measured continuously for 10 h to fully analyze the swelling behavior of the hydrogels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Biodegradation Behavior of Hydrogel\u003c/h2\u003e \u003cp\u003eAnhydrous hydrogels after freeze-drying were weighed (W\u003csub\u003e0\u003c/sub\u003e) and immersed in PBS buffer (pH\u0026thinsp;=\u0026thinsp;7.4) with 0.4 mg/mL of lysozyme (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e U\u0026middot;mg) at 37 ℃. The hydrogels were taken out and weighed (W\u003csub\u003es\u003c/sub\u003e) at different times. The experiment was measured continuously for 6 d to fully analyze the biodegradation behavior of the hydrogels. The degradation rates were calculated using the following formula:\u003c/p\u003e \u003cp\u003eThe degradation rate = (1 \u0026ndash; W\u003csub\u003es\u003c/sub\u003e/W\u003csub\u003e0\u003c/sub\u003e) \u0026times; 100%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.8 In vitro Drug Release\u003c/h2\u003e \u003cp\u003eThe in \u003cem\u003evitro\u003c/em\u003e drug release behavior of the pH/ultrasound dual-responsive hydrogel were investigated using methylene blue (MB) as the model drug. First, a range of concentrations of MB from 0 to 25 \u0026micro;g/mL were prepared. And the absorbance was measured by UV-visible spectroscopy (INSEA, China). The peak absorbance at 465 nm were recorded to fit the calibration curve of MP. And then, 20 \u0026micro;L of MB solution (1.0 mg/mL) was added into the 980 \u0026micro;L of the uncross-linked hydrogel solution to prepare MB-loaded hydrogel (20 \u0026micro;g MB per 1.0 mL hydrogel). The hydrogel of free releasing was set as control. The MB-loaded hydrogels were immersed in 5.0 mL PBS buffer (pH 7.4) to study release behavior. And hydrogels of ultrasound treatment, pH treatment (immersing into pH 6.0 PBS buffer), ultrasound\u0026thinsp;+\u0026thinsp;pH treatment were set as experimental group. The ultrasound treatment was set at 60th, 120th, 180th, and 300th min, and each ultrasound treatment lasted 10 min. The absorbance of MB in the were measured by UV-visible spectroscopy and the released percentages were calculated by the fitted calibration curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Cell Acquisition and Culture\u003c/h2\u003e \u003cp\u003eAs previously described\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, human umbilical cord mesenchymal stem cells (Cyagen, HUXUC-01001, China) were purchased from Cyagen Biotechnology Company. The HUC-MSCs were cultured in MesenCult\u0026trade; MSC Basal Medium (Stemcell Technologies, RC200133, China). The multilineage differentiation potential of HUC-MSCs was verified by inducing osteogenic, chondrogenic, and adipogenic differentiation using differentiation media. Surface markers of HUC-MSCs (CD105, CD73, CD90, CD45, CD34, HLA-DR) were detected using flow cytometry.C2C12 myoblast cell line was purchased from Procella (Procella, CL-0044, China). The cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 \u0026micro;g/mL streptomycin. When the myoblasts reached 70%-80% confluence, they were induced to differentiate into myotubes using DMEM supplemented with 2% horse serum, 100 IU/mL penicillin, and 100 \u0026micro;g/mL streptomycin. RAW cells (RAW 264.7, a murine-derived macrophage cell line) were obtained from the cell bank of the Chinese Academy of Sciences Typical Culture Preservation Committee.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.10 EVs Isolation and Characterization\u003c/h2\u003e \u003cp\u003eThe supernatant from HUC-MSCs culture was collected for EVs isolation using ultracentrifugation. The specific steps were as follows: The collected cell supernatant was centrifuged at 300 g, 4\u0026deg;C for 10 minutes to remove cell debris. The supernatant was then centrifuged at 2000 g, 4\u0026deg;C for 10 minutes to remove dead cell debris. Subsequently, the supernatant was centrifuged at 10,000 g, 4\u0026deg;C for 30 minutes to remove cellular debris. The resulting supernatant was filtered through a 0.22 \u0026micro;m filter and transferred to ultracentrifuge tubes. Ultracentrifugation was performed at 100,000 g (Beckman Coulter, USA) for 70 minutes at 4\u0026deg;C. The pellet obtained after centrifugation was resuspended in 1ml PBS\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Next, Nanosight NS300 system (Malvern, UK) was used for nanoparticle tracking analysis (NTA) to analyze the size distribution and particle concentration of the vesicles. Western blotting was then performed to detect the surface markers CD63, CD81, and CD9 of the EVs (Abcam, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.11 PKH-26 Staining and Cellular Uptake Assay\u003c/h2\u003e \u003cp\u003eThe PKH-26 staining kit (Sigma-Aldrich, D0030, China) was employed to label the EVs. The brief procedure, as provided by the manufacturer, is outlined below: The previously isolated EVs were diluted with Dilution C solution, followed by the addition of 4 \u0026micro;L of PKH-26 dye solution. The mixture was then incubated at room temperature in the dark for 5 minutes. Subsequently, the staining reaction was terminated by adding 500 \u0026micro;L of 1% BSA solution. The labeled EVs were pelleted by centrifugation at 1000000 g for 70 minutes at 4\u0026deg;C, followed by resuspension in 200 \u0026micro;L of cold PBS. C2C12 cells were co-cultured with the labeled EVs for 24 hours. The cells were then fixed with 4% paraformaldehyde, stained with DAPI for 5 minutes to label the cell nuclei, and observed under a fluorescence microscope to visualize the stained cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.12 MiRNA Sequencing Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the tissue using TRIzol\u0026reg; Reagent according the manufacturer\u0026rsquo;s instructions. Then RNA quality was determined by 5300 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). Only high-quality RNA sample was used to construct sequencing library. RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer\u0026rsquo;s instructions (Illumina, San Diego, CA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.13 Proliferation Experiment of C2C12 Cells\u003c/h2\u003e \u003cp\u003eThe proliferation assay of C2C12 cells was conducted using the CCK-8 cell counting kit (CCK-8, Dojindo, Japan) to assess the proliferation of cells stimulated by EVs. In brief, 5000 cells were seeded in a 96-well plate and cultured for 24 hours. Subsequently, 10 \u0026micro;L of CCK-8 solution was added to each well, followed by co-incubation with the cells for 3 hours. Cell proliferation was determined by measuring the absorbance at 450 nm using an enzyme-linked immunosorbent assay plate reader. The data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from three independent replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.14 Immunofluorescence Experiment of C2C12 Cells\u003c/h2\u003e \u003cp\u003eThe immunofluorescence experiment of C2C12 cells (MYOG, DAPI staining) proceeded as follows: After differentiation in high-glucose DMEM medium containing 10% FBS for 3 days, cells in logarithmic growth phase were seeded onto coverslips to form a monolayer. Subsequently, cells were cultured in high-glucose DMEM medium supplemented with 2% horse serum for 7 days. Following this, cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed three times with 1\u0026times; PBS for 5 minutes each. Non-specific sites were then blocked with 1% bovine serum albumin (BSA) at room temperature for 30 minutes. Each coverslip was incubated overnight at 4\u0026deg;C with mouse monoclonal anti-myogenin primary antibody (1:200 dilution in 1% BSA/PBS) (Beyotime, AF7542, China), followed by three washes with 1\u0026times; PBS for 5 minutes each. Subsequently, coverslips were incubated with FITC-conjugated secondary antibody (anti-rabbit or anti-mouse) (1:500 dilution in 1% BSA/PBS) (Beyotime, A0562, China) at room temperature in the dark for more than 1 hour, followed by three washes with 1\u0026times; PBS for 5 minutes each. Finally, coverslips were stained with DAPI (Beyotime, C1005, China) in the dark for 10 minutes, mounted with a mounting medium, and observed and photographed under an immunofluorescence microscope.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.15 Hydrogel's Protective Effect Against ROS in C2C12 Cells\u003c/h2\u003e \u003cp\u003eC2C12 cells were seeded in a plate and treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (400 \u0026micro;mol) for 24 hours to establish an oxidative stress microenvironment. Different concentrations of decellularized extracellular matrix materials were then added, and the cells were co-cultured for 48 h. Cell viability was assessed using the CCK-8 and Calcein-AM/PI dual staining method. Intracellular ROS detection was performed using DCFH-DA as the ROS fluorescent probe. Cells were co-incubated with different treatments for 30 minutes, followed by PBS washing. Fluorescence microscopy was used to observe and image the staining of live/dead cells and ROS. Quantitative analysis was conducted using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.16 Effect of Hydrogel on Macrophage Polarization\u003c/h2\u003e \u003cp\u003eThe expression of the macrophage marker CD86 (M1) in RAW cells was detected using flow cytometry. RAW cells were seeded in a 6-well plate at a density of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL with 2 mL per well. After 12 h, the medium was replaced with hydrogel extract, and cells were cultured for an additional 3 days. Subsequently, the cells were scraped off, washed twice with phosphate-buffered saline (PBS), and thoroughly resuspended in 250 \u0026micro;L of fixation/permeabilization solution, followed by incubation at 4\u0026deg;C for 20 minutes. After washing the cells twice with buffer (from the fixation/permeabilization kit, BD), they were incubated with antibody solution containing CD86 (dilution factor 1:100, H2316, Santa Cruz Biotech) at 4\u0026deg;C for 30 minutes. After antibody incubation, the cells were washed twice with buffer, resuspended in PBS, and analyzed using a flow cytometer (FACS, AriaII, BD).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.17 Animal Model Establishment and Grouping\u003c/h2\u003e \u003cp\u003eThis experiment obtained ethical approval from the Ethics Committee of the Tenth People's Hospital of Shanghai (SHDSYY-2023-3825-3).\u003c/p\u003e \u003cp\u003eThe specific steps of the experiment are as follows: After sodium pentobarbital anesthesia of SD rats, an incision was made on the left hind limb posterior lateral thigh to expose the sciatic nerve, freeing the sciatic nerve while preserving the branches of the sciatic nerve to the thigh muscles. The proximal end of the nerve was freed to the level of the hamstring tendon, and the distal end was carefully freed to the entry points of the nerve branches into the muscles. The left sciatic nerve was cut off at the distal end below the hamstring tendon, and the proximal end of the nerve was inverted and buried in the nearby hamstring muscle belly. Two nylon sutures were used to secure the outer membrane and fascicles of the distal end of the nerve, which was pulled proximally and fixed to the hamstring tendon, ensuring that the distal end of the sciatic nerve was positioned at the distal end of the hamstring tendon. Before closing the wound, the left hind limb was maximally moved to ensure secure fixation of the traction lines. The experimental groups were as follows: control group, surgery only without any treatment; material group, injection of UR-gel immediately after surgery without ultrasound treatment; UR-gel\u0026thinsp;+\u0026thinsp;ultrasound group, injection of UR-gel immediately after surgery, followed by 15 minutes of 1 MHz ultrasound stimulation to the tibialis anterior muscle at the 2nd week, with the remaining treatment the same as before; EVs@UR-gel\u0026thinsp;+\u0026thinsp;ultrasound group, injection of EVs@UR-gel immediately after surgery, with the remaining treatment the same as before.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.18 Measurement of Sciatic Nerve Function Index (SFI)\u003c/h2\u003e \u003cp\u003eA wooden trough with open ends, measuring 60 cm in length, 10 cm in width, and 10 cm in height, was constructed. A piece of white paper weighing 70 g was cut to the same length and width as the trough and laid at the bottom. Rats' hind limbs were colored by dipping them in paint at the ankle joints. The rats were then placed at one end of the trough and allowed to walk towards the other end, leaving 5\u0026ndash;6 footprints on each side. The following six parameters were measured for each clear footprint: ETS (injured toe spread), NTS (normal toe spread), EPL (injured print length), NPL (normal print length), EIT (injured intermediary toe spread), NIT (normal intermediary toe spread).These indices were then input into the Bain formula to calculate the SFI. An SFI of 0 indicates normal function, while \u0026minus;\u0026thinsp;100 indicates complete damage. The Bain formula is as follows:\u003c/p\u003e \u003cp\u003eTSF (toe spread factor) = (ETS-NTS)/NTS;\u003c/p\u003e \u003cp\u003ePLF (print length factor) = (EPL-NPL)/NPL;\u003c/p\u003e \u003cp\u003eITF (intermediary toe spread factor) = (EIT-NIT)/NIT;\u003c/p\u003e \u003cp\u003eSFI\u0026thinsp;=\u0026thinsp;109.5 TSF \u0026minus;\u0026thinsp;38.3 PLF\u0026thinsp;+\u0026thinsp;13.3 ITF \u0026minus;\u0026thinsp;8.8.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.19 Measurement of Maximum Isometric Contraction Force\u003c/h2\u003e \u003cp\u003eA Kocher needle with a diameter of 1.0 mm was used to secure the distal end of the rat's femur and ankle joint to a wooden board, stabilizing the lower leg segment and maintaining muscle length. Simultaneously, the muscle tendon insertion point of the anterior tibial muscle was surgically exposed. The initial stimulation frequency was set at 10 Hz, with a duration of 0.4 milliseconds, and the voltage was set at 2 V. We calibrated the force transducer using weights of 0 g, 10 g, 20 g, 30 g, and 50 g, respectively. The muscle tendon insertion point of the anterior tibial muscle was connected to the force sensor, and a single stimulation of the proximal end of the sciatic nerve anastomosis was applied using the stimulating electrode, maintaining the aforementioned stimulation parameters. We incrementally increased the weight at the muscle tendon insertion point by 0 g, 5 g, 10 g, 15 g, and 20 g increments (increasing by 5 g each time until the optimal length was determined) to find the maximum force generated by this single stimulation at each weight. The muscle length at this point was considered the optimal length. At the optimal length, the voltage was adjusted to 10 V, and the stimulation frequency was varied at 50 Hz, 100 Hz, 150 Hz, and 200 Hz (starting from 50 Hz and increasing by 50 Hz each time) to measure the maximum isometric contraction force under continuous stimulation of the sciatic nerve at equal lengths.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.20 Muscle Circumference, Muscle Wet Weight and Histological Examination\u003c/h2\u003e \u003cp\u003eThe initial length of the muscles at relaxation was measured. Bilateral anterior tibial muscles were excised completely from their origin to insertion points in relaxed muscles. The superficially adherent subcutaneous tissues were carefully removed, then measure the muscle circumference at the thickest part of the muscle and compare it to the unoperated side, and subsequently, the wet weight was measured by using an analytical balance (with a sensitivity of 1 mg, R200D, Germany) and recorded. Frozen sections from the medial heads of the left anterior tibial and gastrocnemius muscles were subjected to Masson's trichrome staining. Acid fuchsin stained muscle fibers red, while aniline blue stained collagen fibers blue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.21 Immunofluorescence Staining of Tissue Sections\u003c/h2\u003e \u003cp\u003eTissue sections from the anterior tibial muscle collected at six weeks post-operation were subjected to immunofluorescence staining. The muscle tissue was fixed in 4% formaldehyde, dehydrated through a sucrose gradient, embedded in OCT compound, and sliced into 12-micrometer sections. These sections were then blocked at room temperature for 1 hour. Subsequently, they were incubated overnight at 4\u0026deg;C with primary antibodies, including mouse anti-MyHC (dilution 1:200, Abcam, Cambridge, UK) and rabbit anti-Col-1 (dilution 1:200, Abcam, Cambridge, UK). After rinsing with PBS, the sections were incubated with the corresponding secondary antibodies at room temperature in the dark for 2 hours. Following another round of PBS washing, the sections were stained with DAPI to visualize the cell nuclei. Finally, fluorescence microscopy was employed for imaging.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e3.22 Transcriptome Sequencing and Bioinformatics Analysis\u003c/h3\u003e\n\u003cp\u003eAt 6th week post-operation, total RNA was extracted from denervated and denervated\u0026thinsp;+\u0026thinsp;EVs@UR-gel anterior tibial muscles using mRNA isolation kit. Subsequently, the RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Ribosomal RNA was enzymatically digested using the TruSeq Stranded Total RNA and Ribo-Zero Gold kits. The fragmented RNA was used as a template for cDNA synthesis and library construction with fragment buffer solution. RNA libraries were then subjected to RNA identification using the Agilent 2100 Bioanalyzer. Sequencing was performed using an Illumina sequencer (HiSeqTM 2500 or Illumina HiSeq X Ten). DESeq software was employed for normalizing mRNA counts for each sample and calculating fold changes. Differential expression of reads between the two groups was assessed using a negative binomial distribution test. Finally, genes with fold changes of either \u0026gt;\u0026thinsp;1.5 or \u0026lt;-1.5, with a q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, were selected as differentially expressed genes. For functional analysis based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, enrichment was considered significant for p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe discovered that the miRNA target genes in HUC-MSCs-EVs are associated with cellular energy metabolism and proliferation pathways, suggesting their potential in preventing denervated muscle atrophy. To efficiently load, protect, and deliver HUC-MSCs-EVs, we developed an ultrasound/pH dual-responsive anti-inflammatory injectable hydrogel. This hydrogel exhibited desirable anti-inflammatory properties and ultrasound controllability. At the early stage of the disease, the hydrogel's pH responsiveness was achieved. The anti-inflammatory and ROS-neutralizing effects of the hydrogel were observed at the initial stage of the disease. At the subsequent stage of muscle atrophy, ultrasound intervention enabled the modifiable release of EVs, which aided in maintaining muscle function and structure after neuromuscular atrophy and enhanced the therapeutic efficacy of HUC-MSCs-sEVs. Overall, the EVs@UR-gel demonstrates significant potential in preventing denervated muscle atrophy and preserving muscle function.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDisclosure of Interest\u003c/h2\u003e \u003cp\u003eThe authors reported no potential conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eZ.B., J.J., and W.L. contributed equally to this work. Z.B., J.J., W.L., F.Z., M.S., J.D., and P.W. designed the research; Z.B., J.J., W.L., J.H., Z.Z., J.H., J.A., J.H., and J.Y. performed the research; F.Z., M.S., J.D., and P.W. analyzed the data; Z.B., J.J., W.L., F.Z., M.S., J.D., and P.W. wrote the paper.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis research was supported by National Natural Science Foundation of China (52222306, 22075212, 21925505 and 22305177), the international scientific collaboration fund of Science and Technology Commission of Shanghai Municipality (21520710100, 23520710900), the fellowship of China Postdoctoral Science Foundation (2022M720107, GZB20230517) and Shanghai\u0026ldquo;Super Postdoc\u0026rdquo; Incentive Plan (2022568), Shanghai Rising-Star Program (Sailing, 23YF1433000), Natural Science Foundation of Shanghai (20ZR1443200). J.D. is the recipient of National Science Fund for Distinguished Young Scholars.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMartin JA et al (2018) Gauging force by tapping tendons. Nat Commun 9:1592\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCisterna BA et al (2020) Active acetylcholine receptors prevent the atrophy of skeletal muscles and favor reinnervation. Nat Commun 11:1073\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCieza A et al (2021) Global estimates of the need for rehabilitation based on the Global Burden of Disease study 2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet 396:2006\u0026ndash;2017\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteinmetz JD et al (2024) Global, regional, and national burden of disorders affecting the nervous system, 1990\u0026ndash;2021: a systematic analysis for the Global Burden of Disease Study 2021. 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J Extracell Vesicles 4:27031\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Denervated muscle atrophy, extracellular vesicles, Hydrogel, Ultrasound Responsiveness","lastPublishedDoi":"10.21203/rs.3.rs-4715142/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4715142/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDenervated muscle atrophy is a common complication following nerve injury, which often leads to irreversible muscle fibrosis due to low treatment efficiency. Recently, bioactive substances such as extracellular vesicles (EVs) have been emerging as an effective therapeutic modality for muscle atrophy. However, the complicated microenvironments of denervated muscle atrophy could reduce the delivery efficiency and even result in the deactivation of EVs. To meet this challenge, an ultrasound and pH-responsive anti-inflammatory injectable hydrogel was developed, which can effectively load and deliver stem cells derived EVs with satisfactory treatment outcomes of denervated muscle atrophy. Carboxymethyl chitosan, oxidized chondrotin sulfate and cystamine dihydrochloride were crosslinked in situ by Schiff base reaction to form an injectable hydrogel, where the reversible covalent bond would break under ultrasound and acidic environments to promote hydrogel degradation and cargo release. Meanwhile, the hydrogel loaded with EVs isolated from human umbilial cord mesenchymal stem cells(HUC-MSCs) can release EVs in a controlled manner upon facile pH/ultrasound manipulation. The experimental results confirmed that the hydrogel loaded with EVs (EVs@UR-gel) was effective in preserving muscle function. After six weeks nerve reconstruction, the maximum muscle strength which is closely related to muscle function, the muscle circumference, the wet weight, can be restored to 89.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96%, 76.02\u0026thinsp;\u0026plusmn;\u0026thinsp;7.49%, 88.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65% of the healthy state, and the sciatic nerve index (SFI) to -0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, respectively. Overall, this hydrogel provided a new platform to maintain the long-term in vivo bioactivity of EVs, achieve tunable EVs release at the site of denervated muscle atrophy based on the state of disease, and restore the morphology and function of muscle as a promising approach for treating denervated muscle atrophy.\u003c/p\u003e","manuscriptTitle":"Treatment of Denervated Muscle Atrophy by Injectable Dual-responsive Hydrogels Loaded with Extracellular Vesicles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-01 08:08:51","doi":"10.21203/rs.3.rs-4715142/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5565bee5-966f-4eec-8b8a-2e0ee19ded29","owner":[],"postedDate":"August 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35365053,"name":"Physical sciences/Materials science/Biomaterials/Biomedical materials"},{"id":35365054,"name":"Biological sciences/Stem cells/Regeneration"},{"id":35365055,"name":"Biological sciences/Biotechnology/Biomaterials/Biomedical materials"},{"id":35365056,"name":"Physical sciences/Chemistry/Polymer chemistry/Biopolymers"}],"tags":[],"updatedAt":"2024-08-28T19:55:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-01 08:08:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4715142","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4715142","identity":"rs-4715142","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}