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

Abstract

Denervated muscle atrophy, a common outcome of nerve injury, often results in irreversible fibrosis due to the limited effectiveness of current therapeutic interventions. While extracellular vesicles (EVs) offer promise for treating muscle atrophy, their therapeutic potential is hindered by challenges in delivery and bioactivity within the complex microenvironment of the injury site. To address this issue, an injectable hydrogel is developed that is responsive to both ultrasound and pH, with inherent anti-inflammatory and antioxidant properties, designed to improve the targeted delivery of stem cell-derived EVs. This hydrogel system allows for controlled release of EVs from human umbilical cord mesenchymal stem cells (HUC-MSCs), adapting to the specific conditions of the injury environment. In vivo studies using a rat model of nerve injury demonstrated that the EV-loaded hydrogel (EVs@UR-gel) significantly preserved muscle function. Six weeks post-nerve reconstruction, treated rats exhibited muscle strength, circumference, and wet weight reaching 89.53 ± 0.96%, 76.02 ± 7.49%, and 88.0 ± 2.65% of healthy controls, respectively, alongside an improvement in the sciatic nerve index (-0.11 ± 0.09). This platform presents a novel therapeutic approach by maintaining EV bioactivity, enabling tunable release based on the disease state, and facilitating the restoration of muscle structure and function.

Keywords: denervated muscle atrophy; extracellular vesicles; hydrogel; ultrasound responsiveness.

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. b) The 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 facilitates 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 muscle atrophy. 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. (Created with bioRender.com).

Figure 1

Characterizations and ultrasound‐responsiveness study of UR‐gel. a) FTIR spectra of OCS, CS, CMCS, and UR‐gel. b‐c) The SEM images of UR‐gel (b) before ultrasound stimulation and (c) after ultrasound stimulation. d) Rheological property of hydrogels with different treatments. e) Swelling ratio of ultrasonic treated UR‐gel and UR‐gel immersed in PBS buffer (pH 7.4). f) Degradation rate of ultrasonic treated UR‐gel and UR‐gel in PBS buffer containing lysozyme. g) The demonstration of injectable properties. h) Optical images of the UR‐gel and Dox‐loaded UR‐gel. i) Optical images of the UR‐gel and DOX‐loaded UR‐gel under ultraviolet light. j) The drug release behavior of UR‐gel under different conditions, and the accelerated release was observed at each ultrasound‐stimulation time point.

Figure 2

EVs extracted from HUC‐MSC have high biological activity to promote the migration, proliferation, and differentiation of C2C12 cells. a‐b) (a)TEM image and (b) NTA result of EVs, scale bar = 100 nm. c) The typical protein (CD81, CD63, CD9) markers analysis of EVs with HUC‐MSC cells as control. d) Sequencing results of miRNA within EVs. e) Representative bright field image and fluorescence micrograph of PKH‐26 (red) labeled HUC‐MSC‐EVs internalized by C2C12 cells, scale bar = 50 µm. f) Transwell experiment of EVs on C2C12 cell migration, scale bar = 150 µm. g) Analysis of the quantity of C2C12 cell migration influenced by EVs. h) Experiment showing the effect of EVs on cell proliferation, scale bar = 50 µm. i) Cell proliferation assay using CCK8 influenced by EVs. j) Experiment demonstrating the promotion of C2C12 cell differentiation by EVs, scale bar = 100 µm (blue represents DAPI staining, green represents MYOG staining). k) Fusion index of C2C12 cells (the percentage of nuclei within MYOG+ cells containing ≥2 nuclei). **p < 0.01 versus the control; ***p < 0.001 versus the control. Data are presented as the mean ± standard.

Figure 3

EVs@UR‐gel showed high biological activity in vitro. a) Growth status of C2C12 cells on the scaffold after seeded for 3 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 = 100 µm. b) Number of cells co‐cultured with the scaffold. c) Depth of cell growth on the scaffold. d) Transwell experiment of C2C12 cells with different approaches. e) Differentiation effect of C2C12 cells with different approaches. f) Activity of cells co‐cultured with different approaches. g) Quantity of C2C12 cell migration. h) The fusion index of C2C12 with different approaches. *p < 0.05 versus the US group, **p < 0.01 versus the US group, ***p < 0.001 versus the US group. Data are presented as the mean ± standard.

Figure 4

UR‐gel has anti‐inflammatory and antioxidant abilities. a) 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 EVs@UR‐gel had a protective effect on C2C12 cell death induced by H₂O₂, scale bar = 100 µm. b) Cell viability assay after co‐cultured with H₂O₂ for 24 h. c) Intracellular ROS scavenging ability of EVs@UR‐gel in H₂O₂‐induced C2C12 using DCFH‐DA as a fluorescent ROS probe, scale bar = 100 µm. d) Intensity of ROS fluorescence. e) After being incubated with 100 ng mL⁻¹ LPS for 24 h beforehand, RAW 264.7 cells treated with PBS, UR‐gel, UR‐gel+US and EVs@ UR‐gel, and analyzed by immunofluorescent staining of CD86, scale bar = 100 µm. f) flow cytometry of CD86 expression. g) Schematic diagram of the gel immune regulation process. (Created with bioRender.com). **p < 0.01 versus the control. Data are presented as the mean ± standard.

Figure 5

EVs@UR‐gel promotes the recovery of motor function. a) Photograph of tibialis anterior muscle from rats after treatment with various strategies for 6 weeks and 12 weeks in different groups. b) Proportion of maximal muscle tension retained in the tibialis anterior muscle of rats. c Diameter of the tibialis anterior muscle in rats. d) Wet weight of the tibialis anterior muscle in rats. e) SFI analysis of rats at 12 weeks. f–g) Numerical analysis related to SFI in rats. *p < 0.05 versus the control; **p < 0.01 versus the control; ***p < 0.001 versus the control. Data are presented as the mean ± standard.

Figure 6

Effects of EVs@UR‐gel on muscle regeneration and type I collagen deposition in target muscle in vivo. a) 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, scale bar = 100 µm. b‐d) The semi‐quantification of (b) numbers of myofibers, (c) MyHC and (d) Col‐1 immunofluorescence signal intensity. e‐f) Go analysis of changes in transcriptome profile of target muscle treated by EVs@UR‐gel+US. group and the control group ‐in (e) up‐ and (f) down‐ regulated genes. g) Differentially expressed genes (DEGs) analysis between the EVs@UR‐gel+US. group and the control group. h) GO Chord plot of interested GO terms and the corresponding DEGs.