Poor wound healing is a significant global health concern, affecting millions annually and placing a substantial burden on healthcare systems. The high cost associated with managing chronic wounds, especially surgical wounds, underscores the need for improved treatment strategies. Delayed wound closure, keloid formation, and hypertrophic scarring can result from disruptions in the normal inflammatory and hypoxic patterns during healing. Effective management of hypoxia and inflammation is crucial for developing next-generation wound dressings. Adipose-derived stem cell (ADSC)-derived exosomes hold promise in tissue regeneration due to their ability to carry therapeutic molecules like growth factors and microRNAs, which exert anti-inflammatory, anti-apoptotic, angiogenic, and cell-proliferative effects. Exosome-loaded hydrogels show potential as multifunctional wound dressings, mitigating oxidative stress, stimulating angiogenesis, and enhancing fibroblast migration. Exosomes offer advantages over stem cells due to their safety and reduced risk of tumorigenicity and embolism. However, a key limitation is the reduced intracellular cargo delivery efficiency of exosomes under hypoxic conditions due to hypoxia-induced endocytic recycling. This study aims to overcome this limitation by developing a novel wound dressing that combines the therapeutic potential of ADSC-derived exosomes with an oxygen-carrying nanobubble system embedded in a self-healing hydrogel. The oxygen nanobubbles (ONBs) are formed by encapsulating oxygen within a glycosylated protein conjugate, enhancing stability and providing free radical scavenging properties. Exosomes are then coated onto these ONBs, creating exosome-coated oxygen nanobubbles (EBOs). The self-healing polyvinyl alcohol/gelatin (PVA/GA) hydrogel provides hemostasis, shape adaptability, and additional antioxidant properties through borate bonds that react with hydrogen peroxide, thereby mitigating inflammation.

The literature extensively supports the use of ADSC-derived exosomes in tissue regeneration. Studies have demonstrated their anti-inflammatory, anti-apoptotic, and angiogenic properties, contributing to enhanced cell migration and proliferation. Exosome-loaded hydrogels have shown promise in accelerating wound healing, both acute and chronic. Several studies highlight the importance of mitigating oxidative stress and stimulating angiogenesis for effective wound healing. However, the challenge of inefficient exosome delivery in hypoxic wound environments has been identified, necessitating the development of strategies to enhance delivery under such conditions. Existing literature on oxygen-carrying nanobubbles and self-healing hydrogels supports their incorporation into a multifunctional wound dressing.

This study involved several key steps: 1. **ADSC Isolation and Exosome Extraction:** ADSCs were isolated from human adipose tissue and cultured. Exosomes were extracted from the conditioned media using ultracentrifugation and characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). 2. **Oxygen Nanobubble (ONB) and Exosome-Coated Oxygen Nanobubble (EBO) Synthesis:** ONBs were synthesized by conjugating BSA to dextran sulfate and encapsulating nanoscale oxygen bubbles via ultrasonication. EBOs were then created by coating ONBs with exosomes using ultrasonication. 3. **Hydrogel Synthesis and Characterization:** A self-healing PVA/GA hydrogel was prepared by mixing PVA, gelatin, and borax. Rheological properties, self-healing capacity, adhesion, and degradation were characterized. The EBOs were incorporated into the hydrogel to create EBO-Gel. 4. **In vitro Studies:** Various in vitro assays were conducted to assess oxygen release, antioxidant properties, exosome delivery enhancement, hemostatic properties, cell viability, proliferation, migration, and angiogenesis using HDF-a cells and HUVECs. Techniques included immunofluorescence, flow cytometry, MTT assay, scratch wound healing assay, transwell migration assay, and tube formation assay. 5. **In vivo Wound Healing Study:** A rat full-thickness wound model was used to evaluate the in vivo efficacy of EBO-Gel compared to control groups (Tegaderm, Blank-Gel, Exo-Gel, ONB-Gel). Wound closure, inflammation, angiogenesis, and collagen deposition were assessed using digital photography, H&E staining, Masson's trichrome staining, and immunofluorescence staining (CD31, DHE, CD86, CD206, IL-6).

The key findings of this study include: 1. **Successful Synthesis and Characterization of EBOs:** The EBOs were successfully synthesized with a core-shell structure, as confirmed by TEM and SEM. NTA and DLS analyses determined their size and zeta potential. 2. **Self-Healing and Adhesive Hydrogel:** The PVA/GA hydrogel demonstrated excellent self-healing and adhesive properties, suitable for irregular and bleeding wounds. Rheological testing showed shear-thinning behavior, making it injectable. 3. **Oxygen Release and Antioxidant Properties:** EBO-Gel effectively released oxygen and scavenged H2O2, mitigating hypoxia and inflammation. In vitro assays showed reduced ROS levels in cells treated with EBO-Gel. 4. **Enhanced Exosome Delivery:** EBO-Gel significantly enhanced exosome delivery into cells under hypoxic conditions, improving the intracellular delivery of exosome cargo compared to exosomes alone. 5. **Hemostatic Properties:** EBO-Gel exhibited strong hemostatic properties both in vitro and in vivo, effectively controlling bleeding in a rat liver hemorrhage model. 6. **In vitro Promotion of Cell Proliferation, Migration, and Angiogenesis:** EBO-Gel significantly increased HDF-a cell proliferation and migration in vitro, and promoted angiogenesis in HUVECs. 7. **Accelerated Wound Healing in vivo:** In the rat full-thickness wound model, EBO-Gel demonstrated significantly accelerated wound healing compared to controls. Histological analysis showed enhanced angiogenesis, reduced inflammation, increased collagen deposition, and improved epidermal and dermal thickness in the EBO-Gel treated group. 8. **Improved Healing Quality:** EBO-Gel resulted in significantly reduced scar index, suggesting improved healing quality and potentially scarless healing.

The results demonstrate that the EBO-Gel effectively addresses multiple challenges in wound healing. The combination of oxygen delivery, enhanced exosome delivery, hemostasis, and anti-inflammatory properties synergistically accelerates wound healing. The in vivo results confirm the superior efficacy of EBO-Gel in promoting wound closure, reducing inflammation, and improving overall healing quality. The observed improvement in angiogenesis and collagen deposition contributes to the faster and better-quality healing. The ability of the hydrogel to mitigate hypoxia is particularly important, as hypoxia significantly impairs the delivery efficacy of exosomes. The reduction in scar index suggests a potential for scarless healing. These findings support the potential of EBO-Gel as an advanced wound dressing for both acute and chronic wounds.

This study successfully developed a multifunctional EBO-Gel for enhanced wound healing. The combination of oxygen nanobubbles, exosomes, and a self-healing hydrogel provided hemostasis, reduced inflammation, mitigated hypoxia, and enhanced exosome delivery, leading to significantly faster and better-quality wound healing in a rat model. Future studies could explore the use of EBO-Gel in treating chronic wounds and other ischemic conditions, and optimize the formulation for different wound types.

The study utilized a rat model, and the results may not fully translate to human conditions. The long-term effects of EBO-Gel and its potential for side effects require further investigation. Further studies with larger sample sizes are needed to confirm the findings.