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• Acute liver failure (ALF) is a critical disease characterized by extensive hepatocyte necrosis, rapid deterioration of liver function, and frequent progression to multi-organ failure, leading to a high mortality rate of up to 40% [1–3]. It is reported that the excessive production and accumulation of reactive oxygen species (ROS) are early pathological markers of ALF, driving disease progression by inducing oxidative stress, inflammatory response, and extensive hepatocyte necrosis [4,5]. Consequently, suppressing inflammation, scavenging overexpressed ROS, and promoting hepatocyte proliferation are primary alternative approaches for ALF treatment.

  In recent years, the development of stem cell-related technologies has opened new avenues in regenerative medicine [6–8]. mesenchymal stem cells (MSCs), for instance, can promote cell regeneration through multiple mechanisms such as differentiation and secretion of regulatory factors [9,10]. Stem cells and their derivatives have been widely applied in various fields of tissue regeneration and repair, including anti-aging, neural regeneration, and vascular reconstruction [11–13]. Similarly, biomaterials have been explored extensively in regenerative medicine and tissue engineering due to their excellent biocompatibility, local injectability, and morphological plasticity [14,15]. Applications include wound dressings, embolic agents, gingival gels, bone and joint fillers, vascular reconstruction, ureter reconstruction, and more [16–19]. Many previous studies have reported that stem cell-derived exosomal vesicles (EV) are rich in various components, including microRNAs (miRNAs), peroxidases, superoxide dismutase (SOD), glutathione peroxidase, anti-inflammatory factor interleukin-10 (IL-10), heat shock proteins (HSPs), and nuclear factor-E2-related factor 2 (Nrf2). These components can regulate intracellular antioxidant and anti-inflammatory responses, protecting cells from inflammation and oxidative stress-induced damage [20–23].

  Significant progress has been made with hydrogels in the field of liver disease [24–26]. For example, Zheng et al. used hydrogels to encapsulate stem cell-derived cytokines and cover surgical wounds after partial hepatectomy, achieving hemostasis and anti-infection effects while greatly promoting liver regeneration and stabilizing liver function during the acute 48-h period [27]. During this acute regenerative phase, hepatocyte growth factor (HGF) effectively promotes liver regeneration by binding to the C-Met receptor on the hepatocyte membrane. Some studies have used nucleoside-modified, lipid nanoparticle-encapsulated mRNA (mRNA-LNP) delivery to transiently express HGF in mice, which effectively induces hepatocyte proliferation. However, the production cost of mRNA is prohibitively high, making clinical application challenging [28].

  Building on the significant contributions of hydrogels and stem cell derivatives in biomedicine [29,30], as well as the efficient regenerative effects of HGF, a hydrogel coating, CCO/HGF@EV, was designed to combine all three (Fig. 1, CCO/HGF@EV synthesis method and material description are in Supporting information).

  Figure 1

  

  Figure 1.  Synthesis pathway and therapeutic mechanism of CCO/HGF@EV.

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  Subsequently, we validated the properties of CCO/HGF@EV through a series of cell and animal experiments. The Ethics Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine has sanctioned our study. This work has been reported in accordance with the ARRIVE guidelines (Animals in Research: Reporting of In Vivo Experiments).

  To prepare a multifunctional dressing that regulates the inflammatory microenvironment at ALF sites, the polysaccharide-based hydrogels (CCO) were designed and synthesized, including chitosan modified with hydrocaffeic acid (CS-CA), oxidized yeast beta-glucan (OBG) and HGF@EV (Fig. S1 in Supporting information). The CS-CA was synthesized via the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) catalyzed amidation between the carboxyl group of CA and amino groups of CS. The obtained CS-CA easily dissolved in phosphate-buffered saline (PBS, pH 7.4) exhibiting a catechol substitution degree of 8.9%, as determined by ultraviolet-visible (UV–vis) spectroscopy, with a characteristic absorption peak at 282 nm (Fig. 2A). In addition, the chemical shift at 6.6–6.9 ppm in the ¹H nuclear magnetic resonance (NMR) spectrum showed a chemical shift of CA, which further proved that confirm the successful formation of CS-CA (Fig. S2 in Supporting information).

  Figure 2

  

  Figure 2.  Characterization of CCO hydrogels. (A) UV–vis of CS and CS-CA. (B) ¹H NMR of BG and OBG. (C) FTIR of BG and OBG. (D) The image of CCO hydrogel. (E) The SEM images of CCO-1 hydrogel and EDS mapping. Scale bar: 30 µm (n = 3). (F) The different pore size of CCO hydrogel. (G–I) The porosity, remaining weight and swelling rate of CCO hydrogel (n = 3). Data are presented as mean ± standard deviation (SD) and analyzed by Mann–Whitney U test or one-way ANOVA. P < 0.05, **P < 0.01.

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  The OBG could be obtained by sodium periodate oxidation. According to the ¹H NMR of beta-glucan (BG) and OBG (Fig. 2B), BG was resoundingly modified with aldehyde groups. Besides, the stretching vibration of C=O could be clearly recognized (1633 cm⁻¹) in the Fourier transform infrared (FTIR) spectrum and the GPC of OBG was analyzed (Fig. 2C, Fig. S3 in Supporting information). By hydroxylamine titration, the aldehyde modification rate was determined to be 35.6%. Then, a tissue-adhesion hydrogel (CCO) was constructed in situ by simply mixing the equal volume of CS-CA and OBG solution, primarily via Schiff base reaction between the amino groups of CS-CA and aldehyde groups of OBG (Fig. 2D).

  As presented in Fig. 2E, we could see the internal morphology of the hydrogel, occurring a porous structure and uniform distribution of elements (C, N, O), which was conducive to swelling and cell adhesion. The image J was used to calculate the pore sizes of hydrogels. The pore sizes of CCO-1, CCO-2 and CCO-3 were 36.9 ± 3.2, 26.7 ± 2.3 and 17.6 ± 2.0 µm, respectively, which may be due to high solid content and crosslinking degree (Fig. 2F). It is noteworthy that increasing OBG concentration gradually impacts the pore size and porosity of the hydrogel. Meanwhile, the CCO hydrogel demonstrated high porosity (50%–70%), enhancing its swelling capacity and suitability as cell carrier (Fig. 2G). As shown in Fig. 2H, after 7 days of degradation, the remaining mass of the three hydrogels were 27.13%, 35.3% and 39.4%, respectively, and the degradation time met the treatment time of liver failure. In addition, we also characterized the swelling rate of the hydrogel. After 48 h treatment, the swelling rate of the hydrogel can reach 230%–320%, and the high swelling rate can maintain structural integrity by preventing the water loss and the pore collapse (Fig. 2I).

  Therefore, to evaluate the self-healing properties of the CCO hydrogels, angular frequency, strain amplitude sweeps measurements at 25 ℃ were conducted on the hydrogel and the rheological behavior upon external strains was studied. As shown in Fig. S4A (Supporting information), the energy storage modulus of the hydrogel was higher than the loss modulus, which indicated that the hydrogel network was uniform gel state. At the same time, the network state of the hydrogel was damaged when different strains were changed. When strain was at 110%, the energy storage modulus of the hydrogel began to be lower than the loss modulus, and the hydrogel changed to the sol state (Fig. S4B in Supporting information). In addition, we further characterized the self-healing characteristics of the hydrogel by rheology. When the strain was 1%, the hydrogel presented uniform characteristics of the hydrogel network; when the strain was 500%, the hydrogel network was destroyed, and the sol state appeared. When the strain was restored to 1%, the modulus of hydrogel basically returned to the original state, and the process lasted for 3 cycles (Fig. S4C in Supporting information), and two different colored hydrogels were able to fuse together on their own after 30 min, which indicated CCO hydrogels were obtained by dynamic Schiff base crosslinking, presenting excellent self-healing characteristics (Fig. S4F in Supporting information). More importantly, CCO hydrogels showed excellent injectability and could obtain uniform lines and different shapes through 16 G syringes, showing strong shear thinning and shape plasticity (Figs. S4D and G in Supporting information). For the tissue adhesion, the hydrogel can maintain an adhesion strength of 18–26 kPa on pig skin tissue, with CCO-2 and CCO-3 showing superior adhesion even in high protein medium environment, correlating with higher solid content and crosslinking degree (Figs. S4E and S5 in Supporting information) [31,32]. In Figs. S4H and I (Supporting information), it can be clearly seen that the hydrogel can maintain good tissue adhesion in gloves and continuous bending deformation as well as on pig skin, indicating that the hydrogel has good mechanical properties and can meet the adhesion requirements for biomaterials.

  After extracting the exosomes, we used scanning electron microscope (SEM) to observe their morphology, which exhibited a distinct cup-shaped structure (Fig. S4J in Supporting information). Through nanoparticle tracking analysis, we observed that the extracted exosomes maintained Brownian motion, with a total concentration reaching 1.6 × 10⁹/mL. Most exosomes were in the 100–150 nm range, with the peak showing that more than half of the exosome particles had a diameter of 128.8 nm, and the average particle size was 165.6 nm (Fig. S4K in Supporting information). Western blot analysis confirmed the high expression of characteristic exosome markers CD9, CD63, syntenin, and Alix. In addition, HGF protein expression in HGF@EV was much higher than that in the EV group, indicating that HGF had been successfully loaded into EV (Fig. S4L in Supporting information), and its encapsulation rate was detected to be about 13.4% using the bicinchoninic acid assay (BCA) method.

  To verify the sustained release of HGF@EV from the CCO hydrogel, we used nanoparticle tracking analysis (NTA) to monitor the release profile. The results showed that HGF@EV was continuously released over 7 days, with > 80% being released from the CCO hydrogel within the first 3 days (Fig. S4M in Supporting information).

  To assess the biocompatibility of EV, HGF@EV, CCO, CCO/EV, and CCO/HGF@EV, we co-cultured these components with the AML12 cell line and used the CCK8 assay to measure AML12 cell viability. At both 24- and 48-h time points, each component was observed to promote AML12 cell proliferation to a certain extent, with HGF@EV and CCO/HGF@EV showing particularly significant proliferation effects (Fig. S6A in Supporting information). To further confirm the safety and cell proliferation-promoting effects of HGF@EV and CCO/HGF@EV, we used live/dead staining to quantitatively assess the survival rate of AML12 cells cultured with or without hydrogels. Fig. S6B (Supporting information) shows the experimental results, where cells cultured with HGF@EV and CCO/HGF@EV extracts for 48 h exhibited marked cell proliferation, with enhanced cell growth and increased cell density. Therefore, we identified HGF@EV and CCO/HGF@EV as highly effective components, and we will compare the other effects of these two groups in subsequent experiments.

  We injected the hydrogel around the liver using ultrasound-guided localization. Through in vivo imaging, we observed that the hydrogel adhered to and enveloped the liver surface, gradually degrading over 3 days. After 72 h, a small amount of hydrogel still remained (Fig. S6C in Supporting information). By the 4^th day, upon opening the abdominal cavity, we found that the hydrogel had completely degraded. The overall morphology of the liver remained intact, and no pathological changes were observed in the major organs of the mice, including the heart, liver, spleen, lungs, and kidneys (Fig. S6D in Supporting information). Additionally, we tested liver function biochemical indicators and found that alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT) and albumin (ALB) showed no significant changes (Fig. S6E in Supporting information). These findings collectively indicate that the CCO/HGF@EV precursor materials, composed of natural biological extracts, have good biocompatibility and do not adversely affect the integrity of cells or organ tissues.

  Due to the accumulation of oxygen free radicals and oxidative damage in liver tissue during the pathophysiological process of acute liver injury, eliminating oxygen free radicals to reduce oxidative damage is a crucial step in treating tissue damage. To evaluate the antioxidant capacity of various hydrogels, in vitro ROS scavenging assays were conducted. The ROS accumulation in HGF@EV and CCO/HGF@EV gradually decreased, with the fluorescence signal in the CCO/HGF@EV group becoming extremely weak. These experimental results demonstrated that CCO/HGF@EV possess excellent ROS scavenging abilities (Figs. S7A and B in Supporting information). Additionally, the death rate of AML12 cells treated with treated with H₂O₂ (after CCO/HGF@EV pretreatment) significantly decreased, approaching that of the Ctrl (−) group (Figs. S7C and D in Supporting information). Subsequently, we used Annexin V/7-aminoactinomycin D (7-AAD) staining to observe AML12 cell lines, and consistent with the live/dead staining results, the proportion of apoptotic cells in the Ctrl (+) group exceeded 70%, while the proportion of apoptotic cells in the HGF@EV group decreased by nearly 20%, and in the CCO/HGF@EV group, it decreased by about half. These results further confirm that CCO/HGF@EV has a strong ROS scavenging ability and powerful anti-apoptosis function in tissues (Figs. S7E and F in Supporting information).

  Through scratch assays (Figs. S8A and B in Supporting information), after co-culturing for 48 h, the migration rate of human umbilical vein endothelial cells (HUVECs) in the HGF@EV and CCO/HGF@EV groups significantly increased, respectively. We determined that CCO/HGF@EV can greatly enhance the migration ability of HUVECs.

  Furthermore, we used tube formation assays to evaluate whether the hydrogel promotes vascular endothelial cell angiogenesis. Our observations indicated that CCO/HGF@EV significantly promoted the rapid proliferation and crosslinking of HUVECs, demonstrating its potential in enhancing tissue repair and angiogenesis (Figs. S8C and D in Supporting information).

  To further verify the regenerative capability of CCO/HGF@EV, we used 5-ethynyl-2′-deoxyuridine (EdU) staining to assess hepatocyte regeneration levels. Consistent with the previous results, the signal intensity of EdU in the CCO/HGF@EV and HGF@EV groups was significantly higher than in the control group (Figs. S8E and F in Supporting information). These findings indicate the great potential and value of CCO/HGF@EV in regenerative medicine.

  To further validate the efficacy of CCO/HGF@EV, we used an ALF model of C57 mice constructed with carbon tetrachloride to validate the protective effect of CCO/HGF@EV against acute liver injury (Fig. 3A). After 2 days of administration, we measured the liver function indicators in the mice's serum and found that the ALT and AST levels in the CCO/HGF@EV group were significantly lower than those in the Ctrl (+) group, while the serum ALB levels were higher (Fig. 3B), indicating that CCO/HGF@EV enhanced the liver function protective effects of HGF@EV. Hematoxylin and eosin staining (H&E) results of liver tissue showed that the necrotic areas in the livers of mice in the CCO/HGF@EV group were significantly smaller (Figs. 3C and D). Further immunofluorescence staining showed relatively low levels of the apoptosis marker terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) (Figs. 3E and F), and the dihydroethidium (DHE) staining results were consistent with the oxidative damage results of previous in vitro experiments and were much lower than those of the Ctrl (+) group (Figs. 3G and H). The reduction in tumor necrosis factor-alpha (TNF-α) signals indicated that CCO/HGF@EV also had remarkable anti-inflammatory capabilities (Figs. 3I and J). We then used Ki67 staining to observe the regeneration of hepatic parenchymal cells and CD31 staining to observe angiogenesis. Interestingly, both the levels of hepatic parenchymal cell regeneration and vascular proliferation were significantly enhanced (Figs. 3K–N). To further validate the efficacy of CCO/HGF@EV in reprogramming macrophages, we assessed the content of M2 macrophages in the tissue. The results showed a significant increase in M2 macrophages in the CCO/HGF@EV group compared to the Ctrl (+) and HGF@EV groups (Figs. 3O and P). This indicates that CCO/HGF@EV has a pronounced ability to reprogram macrophages, thereby exerting strong anti-inflammatory, antioxidant, and tissue repair effects.

  Figure 3

  

  Figure 3.  Treatment efficiency of acute liver injury by CCO/HGF@EV in vivo. (A) Schematic illustration of the establishment of the animal model and treatment timeline. (B) Changes in liver function-related enzyme levels. (C) Representative pictures of H&E staining of liver tissues and (D) necrosis analysis. Scale bar: 200 µm. (E) Representative immunohistochemical staining figures of apoptosis parameters (scale bar: 200 µm) and (F) quantitative analysis. (G) Representative immunohistochemical staining figures of ROS parameters (scale bar: 200 µm) and (H) quantitative analysis. (I) Representative immunohistochemical staining figures of pro-inflammatory parameters (scale bar: 200 µm) and (J) quantitative analysis. (K, M) Representative immunohistochemical staining figures of neovascularization parameters (scale bar: 200 µm) and (L, N) quantitative analysis. (O) Representative immunohistochemical staining figures of change in macrophage phenotype (scale bar: 75 µm) and (P) quantitative analysis. Data are presented as mean ± SD (n = 4) and analyzed by Mann–Whitney U test or one-way ANOVA. P < 0.05, **P < 0.01, ***P < 0.001. ns, non-significant.

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  Through a series of rigorous experiments, it was demonstrated that using ultrasound guidance to inject the hydrogel around the liver, ensuring complete coverage of the mouse liver surface, significantly reduced pro-inflammatory cytokines and oxidative radicals in the hepatic microenvironment.

  CCO/HGF@EV effectively reduced inflammation and oxidative damage, alleviated cell apoptosis and necrosis, and facilitated tissue regeneration and repair. This was achieved through the synergistic action of stem cell regenerative factors enriched in EV and HGF, which together promoted restoration of liver function homeostasis.

  Declaration of interest statement

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Yingqiang Liang: Methodology. Shilun Li: Methodology. Yixiao Pan: Investigation. Weiyu Zhang: Writing – review & editing. Shupeng Liu: Formal analysis. Yiwen Chen: Supervision. Jiangfeng Hu: Writing – review & editing. Xueliang Zhang: Software. Jun Zhao: Funding acquisition. Zhigang Zheng: Writing – original draft, Conceptualization.

  Acknowledgment

  This work was supported by the Fellowship of China Postdoctoral Science Foundation (No. 2023M732299).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2025.110841.

  
  
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  Figure 1  Synthesis pathway and therapeutic mechanism of CCO/HGF@EV.

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  Figure 2  Characterization of CCO hydrogels. (A) UV–vis of CS and CS-CA. (B) ¹H NMR of BG and OBG. (C) FTIR of BG and OBG. (D) The image of CCO hydrogel. (E) The SEM images of CCO-1 hydrogel and EDS mapping. Scale bar: 30 µm (n = 3). (F) The different pore size of CCO hydrogel. (G–I) The porosity, remaining weight and swelling rate of CCO hydrogel (n = 3). Data are presented as mean ± standard deviation (SD) and analyzed by Mann–Whitney U test or one-way ANOVA. P < 0.05, **P < 0.01.

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  Figure 3  Treatment efficiency of acute liver injury by CCO/HGF@EV in vivo. (A) Schematic illustration of the establishment of the animal model and treatment timeline. (B) Changes in liver function-related enzyme levels. (C) Representative pictures of H&E staining of liver tissues and (D) necrosis analysis. Scale bar: 200 µm. (E) Representative immunohistochemical staining figures of apoptosis parameters (scale bar: 200 µm) and (F) quantitative analysis. (G) Representative immunohistochemical staining figures of ROS parameters (scale bar: 200 µm) and (H) quantitative analysis. (I) Representative immunohistochemical staining figures of pro-inflammatory parameters (scale bar: 200 µm) and (J) quantitative analysis. (K, M) Representative immunohistochemical staining figures of neovascularization parameters (scale bar: 200 µm) and (L, N) quantitative analysis. (O) Representative immunohistochemical staining figures of change in macrophage phenotype (scale bar: 75 µm) and (P) quantitative analysis. Data are presented as mean ± SD (n = 4) and analyzed by Mann–Whitney U test or one-way ANOVA. P < 0.05, **P < 0.01, ***P < 0.001. ns, non-significant.

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