The effect of Adipose-derived stem cells exosomes cross-linked Chitosan-αβ-glycerophosphate thermosensitive hydrogel on deep burn wounds

The effect of Adipose-derived stem cells exosomes cross-linked Chitosan-αβ-glycerophosphate thermosensitive hydrogel on deep burn wounds | 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 Research Article The effect of Adipose-derived stem cells exosomes cross-linked Chitosan-αβ-glycerophosphate thermosensitive hydrogel on deep burn wounds Lei Xu, Dan Liu, Hai Long Yun, Wei Zhang, Li Ren, Wen Wen Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4564135/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 Objective This study aimed to explore the effects on controlling infection and promoting wound healing in deep burn injuries by crosslinking ASCs-Exos with CS-αβ-GP thermosensitive hydrogel. Methods Rats with established deep burn injury models were divided into four groups: CS + ASCs-Exos group, ASCs-Exos group, CS group, and control group. The wound healing rates were analyzed and calculated using Image J software immediately after wound formation and on days 2, 4, 6, 8, 10, 12, and 14 after treatment. Fourteen days after treatment, skin tissues from the wound area, wound margin, and normal full-thickness skin were excised from each group for HE staining and Masson staining. Subsequently, IHC staining was performed on the newly formed wound tissues to detect the expression of TNF-α, IL-6, IL-1α, IL-10, TGF-β, and EGF. Finally, RNA was extracted from the wound tissues, and qPCR was used to detect the mRNA expression levels of IL-1α, CD86, CCL22, and CD163. Results The wound healing rate in the CS + ASCs-Exos group was higher than that in the other groups. HE staining revealed that the CS + ASCs-Exos group had fewer inflammatory cells, a small number of blood vessels, and muscle fibers and collagen fibers distributed alternately in the wound edge at 14 days, which was consistent with normal tissue. Masson staining showed that the wound and wound edge in the CS + ASCs-Exos group at 14 days displayed alternating distributions of collagen fibers and muscle fibers, which was consistent with normal tissue. However, the staining of collagen fibers in the other groups was stronger than that in the experimental group. IHC staining showed that the expressions of IL-10, TGF-β, and EGF in the CS + ASCs-Exos group were slightly higher than those in the other groups, while the expressions of TNF-α, IL-6, IL-1α, and IL-10 were lower than those in the other groups. qPCR detection revealed that the expressions of IL-1α and CD86 in the CS + ASCs-Exos group were lower than those in the control group, while the expressions of CCL22 and CD163 were higher than those in the control group. Conclusion Our research has demonstrated that ASCs-Exos crosslinked with CS-αβ-GP thermosensitive hydrogel exhibits anti-inflammatory properties, promotes wound healing, and enhances the transformation of M1 macrophages into M2 macrophages, with stronger effects compared to ASCs-Exos alone. This provides a new administration method for the clinical application of MSCs-Exos. Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Human skin is the largest organ of the body, whose main function is to provide appropriate protection and prevent external factors and pathogens from invading the body. As wars and natural disasters gradually increase, burns have become a huge challenge affecting skin integrity. Although there have been many studies related to burns, there are still great challenges in the management and treatment of burn wounds. In recent years, with the development of regenerative medicine and tissue engineering, there are endless means for chronic refractory wounds, among which MSCs-Exos(Mesenchymal stem cell exosomes)are expected to become the best measure to solve this problem. A large amount of research evidence indicates that MSCs-Exos can promote the healing of burn wounds, and compared with MSCs, they have significant advantages in terms of storage stability, ease of collection, and safety [ 1 – 3 ]. However, current studies exploring the use of MSCs-Exos for wound repair mainly adopt subcutaneous injection and intravenous injection as the main administration methods, which face the problems of burst release and rapid clearance, making it difficult to play their due role in the complex stages of wound repair. Currently, research is underway to load MSCs-Exos onto patches, injectable microcarriers, or hydrogels to maintain the function of Exos(Exosomes) at the wound site and improve efficiency and safety. Multiple studies have shown that various artificial injectable hydrogels can not only promote the sustained release of Exos in the body but also increase the local retention of Exos [ 4 – 6 ]. These hydrogel dressings include CS (Chitosan), alginate, urea pyridine ketone, polypeptide-based FHE hydrogel, etc., among which CS exhibits high antibacterial activity against fungi, bacteria, algae, and viruses [ 7 ], and creates a moist healing environment for the wound. Therefore, this study was designed to investigate the effect of CS-αβ-GP (Chitosan-αβ-glycerophosphate) thermosensitive hydrogel cross-linked with ASCs-Exos (Adipose-derived stem cells exosomes) on deep burn wounds and verify whether CS-αβ-GP thermosensitive hydrogel is the best carrier for loading MSCs-Exos to promote the healing of deep burn wounds. 2 Materials and Methods 2.1 Isolation and Culture of ASCs-Exos The SD rat ASCs (product code: RASMD-01001) were purchased from OriCell. The 1 mL of cells were aspirated with a sterile pipette into a 5 mL sterile EP tube, diluted with 9 mL of basic medium, and centrifuged at 1000 rpm for 5 minutes to allow the cells to settle. The supernatant was discarded. Four milliliters of freshly prepared medium containing 10% serum were added, gently mixed, and the cells were transferred to a 25T culture flask. The culture flask was placed flat and mixed in a figure-eight pattern before being placed in a 37°C, 5% CO 2 incubator for culture. After 24 hours of culture, the cells were observed under a microscope to ensure adherence, and the medium was carefully replaced for continued culture until the cells reached 90%-100% confluence, at which point the medium was discarded. One milliliter of PBS was added to rinse the cells, repeated three times, and the PBS was discarded. Four milliliters of complete medium were then added. Half of the detached cells were transferred to a new culture flask, and each flask was supplemented with complete medium to 4 mL. The flasks were carefully sealed, placed flat, mixed in a figure-eight pattern, and placed in a 37°C, 5% CO 2 incubator for culture. Once the cells reached confluence, they were passaged using the same method. Cells with good vitality and approximately 70% density were selected for cryopreservation. They were transferred to a sterile EP tube and centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The resulting pellet contained the ASCs-Exos. Freshly prepared cell cryopreservation solution was added, with 2 mL per 25T cell culture, and the solution was divided into two cryopreservation tubes, aiming for a cell density of 5x10 6 -1x10 7 cells per mL. The tubes were placed in a -80°C freezer overnight, and the next day, the cryopreservation tubes were quickly transferred to liquid nitrogen for long-term storage. 2.2 Identification of ASCs-Exos 2.2.1 Electron microscopy observation Cells were taken out from the incubator. The concentration of Exos was adjusted to 0.5g/L, resuspended with PBS, and fixed with 2.5% glutaraldehyde for 10 minutes. 20–30µL of the fixed Exos suspension was taken and dropped onto a carbon-coated copper grid and allowed to stand at room temperature for 1 minute. It was then dried under an infrared lamp for 5 minutes. 1% phosphotungstic acid was added dropwise onto the copper grid and stained at room temperature for 5 minutes. The phosphotungstic acid solution was then absorbed and dried again under an infrared lamp for 10 minutes. Finally, the copper grid was placed under a transmission electron microscope for observation and imaging. 2.2.2 WB (western blot) Detection of Protein Expression Level Cells were taken out from the incubator, and the cell culture medium was discarded. The cells were washed with PBS once. The PBS was discarded, and cell lysate and PMSF were added with a ratio of 100:1. The mixture was placed on ice at 4℃. The cells were fully lysed by pipetting, and the cell lysate samples were transferred to a centrifuge tube and further lysed on ice for 10 ~ 15 minutes. Then, the samples were centrifuged at 12,000 rpm for 10 minutes at 4℃. The supernatant was transferred to a new centrifuge tube, and loading buffer was added. The samples were heated at 100℃ for 20 minutes in a thermostat. After that, the samples were centrifuged at 12,000 rpm for 1 minute at 4℃ and stored at -20℃ for future use. After gel preparation and sample loading, electrophoresis and immunostaining were performed. 2.2.3 The preparation of thermosensitive hydrogel cross-linked ASCs-Exos with CS-αβ-GP Add 2g of CS with 75% deacetylation degree to 100ml of acetic acid/sodium acetate buffer solution with a given pH value of 4.6, and stir until it is completely dissolved. Then, filter out impurities with a 100-mesh cell sieve, sterilize it in a high-pressure sterilizer, and cool it in an ice bath for 20 minutes. Prepare a 50% W/V (weight/volume) αβ-GP aqueous solution in distilled water, sterilize it in a high-pressure sterilizer, and add it dropwise to the CS solution in the ice bath while stirring according to the ratio of CS/αβ-GP at 8.8/1.2. Cool the final mixed CS-αβ-GP solution in the ice bath for 20 minutes, and store it at 4℃ for later use. Mix Exos with the CS-αβ-GP hydrogel to obtain a working solution of CS-Exos with a mass concentration of 100g/L of Exos. After incubating it at 37℃ for 30 minutes, the Exos cross-linked CS-αβ-GP thermo-sensitive hydrogel is formed. 2.4 Animal and Ethical Statement Forty 8-week-old healthy SPF-grade male Sprague Dawley (SD) rats, with body weights ranging from 200 to 220 grams, were purchased from the Experimental Animal Center of Western Theater General Hospital. After purchase, the rats were conventionally kept for one week of adaptation under conditions of (22 ± 2) ℃, 40%-60% humidity, and a 12-hour light/dark cycle, with free access to food and water. The animal study was approved by the Ethics Committee of the General Hospital of the Western Theater Command (Ethics Approval Number: XBZQZYY2021-046). 2.5 Establishment of deep second-degree burn model in rats The rats were anesthetized by intravenous injection of thiopental sodium (40mg/kg) in the tail. After shaving the hair on the back, the skin on the back was scalded with 80℃ water for 8 seconds, with a wound diameter of 16mm. Then, the wound was covered with sterile gauze soaked in physiological saline for 6 minutes. Debridement was not performed on all rats. The rats with deep burns were divided into the following groups: a. CS-ASCs-Exos group: the CS-αβ-GP thermosensitive hydrogel cross-linked with Exos containing 200µg Exos was locally applied to cover the full-thickness skin wound, and a transparent dressing was applied over it. The wound dressing was replaced every 2 days. b. CS group: the hydrogel with the same volume as the CS-αβ-GP thermosensitive hydrogel cross-linked with Exos containing 200µg Exos was locally applied to cover the full-thickness skin wound, and a transparent dressing was applied over it. The wound dressing was replaced every 2 days. c. ASCs-Exos group: 200µg Exos was dissolved in 200µl PBS and locally dispersed onto the full-thickness skin wound using a pipette, and a transparent dressing was applied over it. The wound dressing was replaced every 2 days. d. Control group: only a transparent dressing was locally applied to cover the wound, and it was replaced every 2 days without further treatment. 2.6 Assessment of wound healing Immediately after the formation of wounds and at 2, 4, 6, 8, 10, 12, and 14 days after treatment, the wound healing of rats in each group was observed and recorded. Image J software was used to analyze and calculate the wound healing rate at each time point. The wound healing rate = (1 - unhealed wound area / original wound area) × 100%. 2.7 Pathological assessment of wound healing After 14 days of treatment, full-thickness skin tissues of the wounds and wound edges in the four groups were excised, respectively. Normal full-thickness skin tissues were also excised from the corresponding positions on the dorsal wounds of the rats. After fixation with 40 g/L paraformaldehyde for 24 h, dehydration, paraffin embedding, and slicing (4 µm) were performed successively. Finally, HE(hematoxylin-eosin) staining was used for staining. Masson staining was used to observe the deposition of collagen fibers in the wound tissues. 2.8 Immunohistochemical assessment of wound Immunohistochemical methods were used to detect the expression of TNF-α, TGF-β, IL-1α, IL-6, IL-10, and EGF (Epidermal growth factor) in newly formed wound tissue. TNF-α, IL-1α, and IL-6 were used to observe the proinflammatory status of the wound in rats, while IL-10 and TGF-β were used to observe the anti-inflammatory status of the wound in rats. EGF was used to observe the wound healing status. 2.9 qPCR (Real-time quantitative polymerase chain reaction) Detection of Gene Expression The deep second-degree burn model of rats was constructed according to the modeling method in 2.5. After 14 days of treatment according to the experimental grouping, RNA was extracted from the wound surface and qPCR was used to detect the mRNA expression levels of IL-1α, CD86, CCL22, and CD163. IL-1α and CD86 are markers of M1 macrophages, while CCL22 and CD163 are markers of M2 macrophages. 3 Results 3.1 Characterization of ASCs-Exos To ensure the reliability of subsequent experiments, we first characterized ASCs-Exos. The commercially purchased ASCs cells were cultured, and then 100ml of supernatant from the culture medium was taken for exosome extraction. Transmission electron microscopy was used for observation and identification. As shown in Fig. 1 A, a large number of vesicle-like enrichments with double-concave bilayer membrane structures were observed visually under the electron microscope, with sizes ranging from 30 to 150 nm. Then, 100ml of supernatant from the culture medium of ASCs cells was taken for exosome extraction, and WB was used to detect the expression of typical exosome markers CD9, CD63, and CD81. As shown in Fig. 1 B, after culturing and passage of ASCs cells, CD9, CD63, and CD81 protein expressions were detected in the extracts. These results indicate that the exosome isolation was successful and subsequent experiments can be carried out. 3.2 Accelerating wound healing in deep burn injuries in rats through cross-linking ASCs-Exos with CS-αβ-GP thermosensitive hydrogel To demonstrate that CS-αβ-GP thermosensitive hydrogel is an effective carrier for transporting Exos, we cross-linked ASCs-Exos to CS-αβ-GP thermosensitive hydrogel and applied it to a deep second-degree burn injury model in rats to verify its effect. As shown in Fig. 2 , on days 0, 2, 4, 6, 8, 10, 12, and 14 after wound formation and treatment, the wound healing rate in the CS + ASCs-Exos group was significantly higher from day 4 onward compared to the CS group, ASCs-Exos group, and control group. The final healing rate on day 14 was also higher than the other three groups, indicating that CS + ASCs-Exos can accelerate wound healing and is superior to both the CS hydrogel group and the ASCs-Exos group. 3.3 Reducing Inflammation of Wound Surface after Cross-Linking ASCs-Exos with CS-αβ-GP thermosensitive hydrogel To verify the effect of Exos cross-linked with CS-αβ-GP thermosensitive hydrogel on the wound surface, full-thickness skin tissues of the wound and wound margin from the three groups were resected after 14 days of treatment, and normal full-thickness skin tissues were resected from the corresponding positions on the back of the rats for HE staining and Masson staining. As shown in Fig. 3 A, HE staining revealed that the CS + ASCs-Exos group had a small number of inflammatory cells, a small amount of blood vessels, and muscle and collagen fibers distributed at intervals consistent with normal tissues at the wound margin on day 14, indicating that ASCs-Exos cross-linked with CS-αβ-GP thermosensitive hydrogel can reduce the inflammatory response of the wound surface. As shown in Fig. 3 B, Masson staining showed that the collagen and muscle fibers in the CS + ASCs-Exos group were distributed at intervals in the wound and wound margin on day 14, consistent with normal tissues. In other groups, the staining of collagen fibers was stronger than that in the experimental group, suggesting that the CS + ASCs-Exos group produced less collagen fibers and had a better outcome of scar repair. Then, IHC (immunohistochemical) staining was performed on the skin tissues to detect the expression of TNF-α, IL-1α, IL-6, IL-10, TGF-β, and EGF. The results are shown in Fig. 3 C. The expressions of IL-10, TGF-β, and EGF in the CS + ASCs-Exos group were slightly higher than those in other groups, while the expressions of TNF-α, IL-1α, IL-6, and IL-10 were lower than those in other groups, indicating that CS + ASCs-Exos can increase the anti-inflammatory effect, reduce inflammatory factors, and promote wound healing. 3.4 After crosslinking with CS-αβ-GP thermosensitive hydrogel, ASCs-Exos promote the transformation of M1 macrophages into M2 macrophages. To further verify the effect of Exos crosslinked with CS-αβ-GP thermosensitive hydrogel on macrophages, qPCR was used to detect the mRNA expression levels of M1 macrophage markers IL-1α, CD86, and M2 macrophage markers CCL22, CD163. As shown in Fig. 4 , the expressions of IL-1α and CD86 in the CS + ASCs-Exos group were lower than those in the control group, while the expressions of CCL22 and CD163 were higher than those in the control group, indicating that CS + ASCs-Exos can promote the transformation of M1 macrophages to M2 macrophages, and the transformation effect is stronger compared with the ASCs-Exos group. 4 Discussion MSCs refer to multipotent progenitor cells that can differentiate into osteoblasts, chondrocytes, and adipocytes under standard in vitro differentiation conditions. MSCs are divided into BM-MSCs (Bone-marrow derived stem cells), ASCs (Adipose-derived stem cells), UC-MSCs (Umbilical cord-derived stem cells), iPSC-MSCs (human induced pluripotent stem cell-derived MSCs) and BD-MSCs (Burn tissue-derived mesenchymal stem cells) according to their sources. Among them, ASCs can be obtained from autologous or allogeneic subcutaneous adipose tissues of the abdomen, thighs, and arms through liposuction and separation. Almost 5,000 ASCs can be extracted from 1 gram of adipose tissue, which is easy to obtain and has received increasing attention. Studies have shown that MSCs are recruited to the damaged tissue sites during tissue injury and activated under the close interaction of the inflammatory environment and the immune system [ 8 ]. In response to the inflammatory environment, MSCs secrete some cytokines and growth factors and change the composition of local cytokines, which is considered beneficial to wound healing and tissue regeneration [ 9 ]. Additionally, MSCs have been proven to promote wound healing by increasing angiogenesis, inhibiting inflammation, and promoting fibroblast migration and collagen production through paracrine mechanisms [ 9 ]. All these characteristics are considered crucial for rapid wound healing and are expected to control wound infection, prevent the progression of burn wounds, accelerate skin healing, restore skin barrier function, and reduce scar formation in the treatment of burns. However, MSCs have issues such as tumorigenicity, immune rejection, infection transmission, and complex material storage, which limit their applications. Compared with MSCs, MSCs-Exos have the following advantages: (1) Long-term storage stability: MSCs-Exos are about one millionth of the size of MSCs, have lower complexity, stable structure, are easy to produce and store, and are not affected by storage at -20°C for one week. Their activity can be maintained even after long-term storage at -80°C [ 10 ]. (2) Easy to collect: Various types of MSCs can secrete Exos, and each type can produce 1000 to 10000 of them. Exosomes can be extracted from the culture medium through methods such as ultracentrifugation, and specialized cell lines can also be used to produce Exos on a large scale. Compared with MSCs, the production of MSCs-Exos is simpler, cheaper, and less time-consuming [ 11 ]. (3) Safety: Cell-based therapies using MSCs have issues such as cell survival, regenerative capacity, immune rejection, and tumor differentiation. As a cell-free therapy, Exos can avoid these problems. Due to their low membrane-bound protein content, the possibility of immune rejection is very low even after allogeneic administration. Additionally, Exos do not proliferate, thus eliminating the possibility of tumor formation [ 12 ]. Therefore, MSCs-Exos have better stability, economy, and safety than MSCs in clinical applications. Research on skin wound healing has shown that MSCs-Exos can accelerate skin healing and reduce excessive scar formation. Zhang et al. injected human iPSC-MSCs-Exos subcutaneously around the wound site of a rat model and evaluated the efficacy of human iPSC-MSCs-Exos by measuring wound closure area, histology, and immunofluorescence. The results showed that human iPSC-MSCs-Exos implanted into the wound could accelerate epithelial reformation, reduce scar width, and promote collagen maturation. In addition, it not only promoted the formation of new blood vessels but also accelerated their maturation at the wound site. Furthermore, iPSC-MSCs-Exos stimulated the proliferation and migration of human dermal fibroblasts and human umbilical vein endothelial cells in a dose-dependent manner in vitro and promoted the secretion of type I and III collagen and elastin [ 13 ]. Another study used human UC-MSCs-Exos injected subcutaneously into a rat model with deep second-degree burn wounds on the skin, which showed a significant acceleration of epithelial reformation and increased expression of CK19 (Cytokeratin 19), PCNA (Proliferating cell nuclear antigen), and collagen I (Compared to collagen III) in vivo. In vivo studies confirmed that MSCs-Exos-mediated activation of Wnt/β-catenin could promote wound re-epithelialization and cell proliferation. Knockout of Wnt4 in MSCs-Exos reduced the therapeutic effect in vivo [ 14 ]. Li et al. established a rat model of third-degree burns and intravenously injected human UC-MSCs-Exos. They found that the endogenous miR-181c in MSCs-Exos could inhibit the TLR4 signaling pathway, thereby reducing LPS (Lipopolysaccharide)-mediated inflammation. Administration of human UC-MSCs-Exos overexpressing miR-181c reversed the upregulation of inflammatory factors such as TNF-α and IL-1β and the downregulation of the anti-inflammatory factor IL-10 caused by burns [ 15 ]. Hu et al. studied the role of ASCs-Exos in skin wound healing. The results showed that both local injection and intravenous injection of ASCs-Exos could be taken up and internalized by fibroblasts, stimulating cell migration, proliferation, and collagen synthesis in a dose-dependent manner. It also increased the gene expression of N-cadherin, cyclin-1, PCNA, and collagen I and III. In the early stages of wound healing, systemic administration of ASCs-Exos increased the production of collagen I and III, while in the later stages, it might inhibit collagen expression to reduce scar formation. This suggests that ASCs-Exos can promote skin wound healing by optimizing the characteristics of fibroblasts [ 16 ]. Ma et al. exposed HaCaT cells (Human immortalized keratinocytes) to H2O2 to establish a skin lesion model and then used human ASCs-Exos. They found that ASCs-Exos could promote the proliferation and migration of HaCaT cells and inhibit their apoptosis. In addition, the enhancement of β-catenin at the protein level confirmed the activation of the Wnt/β-catenin signaling pathway, suggesting that ASCs-Exos may promote skin wound healing through Wnt/β-catenin signaling [ 17 ]. Macrophages are divided into M1 and M2 subtypes based on their activation status. M1 macrophages release proinflammatory factors such as TNF-α, IL-1α, IL-1β, IL-6, CXCL9 (C-X-C motif chemokine ligand 9), and CXCL10, while M2 macrophages secrete anti-inflammatory mediators, including IL-10, TGF-β, CCL1 (C-C motif chemokine ligand 1), CCL17, CCL18, and CCL22 [ 18 ]. He et al. intravenously injected human BM-MSCs-Exos into mice with full-thickness skin excision and showed that miR-223 in MSCs-Exos induced macrophage polarization to the M2 subtype by targeting pknox1, thereby accelerating skin wound healing. This demonstrates that miRNAs in MSCs-Exos can be used to promote skin wound healing [ 19 ]. Dalirfardouei et al. established full-thickness excision wounds on the backs of diabetic mice induced by streptozotocin to simulate diabetic foot ulcers. Then, they used menstrual blood-derived MSCs-Exos for subcutaneous injection and observed that MSCs-Exos enhanced neovascularization by upregulating vascular endothelial growth factor A, inhibited inflammation by inducing M1 macrophage polarization to M2, accelerated re-epithelialization, and reduced scar formation by lowering the Col1 (Collagen type 1):Col3 ratio. This suggests that menstrual blood-derived MSCs-Exos can improve the non-healing of diabetic foot ulcers [ 20 ]. Another study using a mouse model of diabetic foot ulcer showed that intravenous injection of BM-MSCs-Exos overexpressing lncRNA H19 prevented the apoptosis and inflammation of fibroblasts by weakening the PTEN (Phosphatase with tensin homology) inhibition mediated by miR-152-3p, thus promoting wound healing in mice with diabetic foot ulcer [ 21 ]. Although a large amount of research evidence suggests that MSCs-Exos can promote the healing of burn wounds, the current main administration methods are subcutaneous injection and intravenous injection, which face the problems of burst release and rapid clearance, which are not conducive to wound healing. Multiple studies have shown that various injectable artificial hydrogels can not only promote the sustained release of Exos in vivo, but also increase the local retention of Exos [ 4 – 6 ]. Hydrogel dressings include CS, alginate, ureido pyrimidinone, peptide-based FHE hydrogel, etc. Among them, CS also shows high antibacterial activity against fungi, bacteria, algae, and viruses, and creates a wet healing environment for the wound [ 7 ]. CS-αβ-GP hydrogel is a flowable viscous liquid at temperatures below 37°C, which turns into a non-flowable gel when heated to 37°C. CS-αβ-GP thermosensitive hydrogel with a pH of 4.6, an ionic strength of 0.15 mol/L, and a CS/αβ-GP ratio of 8.8/1.2 is stable at 4°C for at least 3 months, so it is hopeful to become the best carrier for loading MSCs-Exos [ 22 ]. Our findings reveal that the CS + ASCs-Exos group exhibited a marked enhancement in wound healing rates starting from day 4, ultimately surpassing the healing rates observed in the CS group, ASCs-Exos group, and control group on day 14. This suggests that CS + ASCs-Exos can accelerate wound healing and is superior to the other groups. HE staining revealed that in the CS + ASCs-Exos group on day 14, there were fewer inflammatory cells at the wound margin, with a small number of blood vessels, and the distribution of muscle fibers and collagen fibers was consistent with normal tissue. This suggests that the combination of ASCs-Exos and CS-αβ-GP thermosensitive hydrogel can reduce the inflammatory response at the wound site. Masson staining showed that in the CS + ASCs-Exos group on day 14, the wound and wound margin exhibited a distribution of collagen fibers and muscle fibers consistent with normal tissue. In contrast, the staining of collagen fibers in the other groups was stronger than in the experimental group, indicating that the CS + ASCs-Exos group produced less collagen fiber and had a better outcome of scar repair. IHC staining revealed that the expressions of IL-10, TGF-β, and EGF in the CS + ASCs-Exos group were slightly higher than in the other groups, while the expressions of TNF-α, IL-6, IL-1α, and IL-10 were lower. This suggests that CS + ASCs-Exos can increase anti-inflammatory effects, reduce inflammatory factors, and promote wound healing. qPCR analysis showed that the expressions of IL-1α and CD86 in the CS + ASCs-Exos group were lower than in the control group, while the expressions of CCL22 and CD163 were higher. This indicates that CS + ASCs-Exos can promote the transformation of M1 macrophages to M2 macrophages, and this effect is stronger compared to the ASCs-Exos group. In summary, our study has demonstrated that ASCs-Exos cross-linked CS-αβ-GP thermosensitive hydrogel has anti-inflammatory effects, promotes wound healing, and facilitates the transition of M1 macrophages to M2 macrophages. Moreover, its effects are stronger compared to ASCs-Exos alone. However, there are still some limitations in our research. First, it is unclear how ASCs-Exos cross-linked CS-αβ-GP thermosensitive hydrogel regulates signaling pathways. Second, the molecular substances enriched after ASCs-Exos cross-linked CS-αβ-GP thermosensitive hydrogel remain to be studied. Third, in the current study, we only verified the effects of ASCs-Exos cross-linked CS-αβ-GP thermosensitive hydrogel on deep burn wounds in animal models. Nevertheless, our results provide a new method of administration for the clinical application of MSCs-Exos. Further clinical studies are needed to validate our findings. Declarations Declarations Ethical Approval and consent to participate The animal study was approved by the Ethics Committee of the General Hospital of the Western Theater Command (Ethics Approval Number: XBZQZYY2021-046). Consent for publication: Not applicable. Conflict of interest There are no conflicts of interest. Funding This work was supported by the incubation program of the General Hospital of the Western Theater Command (Nos. 2021-XZYG-C29 and 2021-XZYG-C45). The funders had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript. Author Contribution Han, Xu, Liu, Zhang, Ren and Li wrote the main manuscript text. Xu and Yun prepared Figure 1-4. All authors reviewed the manuscript. Acknowledgement We thank Dr Xian Hui Li from the Department of Burns in General Hospital of the Western Theater Command, and Dr Hao Yao from Department of hematology in General Hospital of the Western Theater Command for their contributions to the study. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request. References Yu B, Zhang X, Li X. Exosomes derived from mesenchymal stem cells. Int J Mol Sci. 2014;15(3):4142–57. Mendt M, Kamerkar S, Sugimoto H, et al. Generation and testing of clinical-grade exosomes for pancreatic cancer. JCI Insight. 2018;3(8):e99263. Marote A, Teixeira FG, Mendes-Pinheiro B, Salgado AJ. MSCs-Derived Exosomes: Cell-Secreted Nanovesicles with Regenerative Potential. Front Pharmacol. 2016;7:231. Shi Q, Qian Z, Liu D, et al. GMSCs-Derived Exosomes Combined with a Chitosan/Silk Hydrogel Sponge Accelerates Wound Healing in a Diabetic Rat Skin Defect Model. Front Physiol. 2017;8:904. Shafei S, Khanmohammadi M, Heidari R, et al. Exosome loaded alginate hydrogel promotes tissue regeneration in full-thickness skin wounds: An in vivo study. J Biomed Mater Res A. 2020;108(3):545–556. Mol EA, Lei Z, Roefs MT, et al. Injectable Supramolecular Ureidopyrimidinone Hydrogels Provide Sustained Release of Extracellular Vesicle Therapeutics. Adv Healthc Mater. 2019;8(20):e1900847. Goy R C, Morais S T B, Assis O B G. Evaluation of the antimicrobial activity of chitosan and its quaternized derivative on E. coli and S. aureus growth[J]. Revista Brasileira de Farmacognosia, 2016, 26: 122–127. Rangatchew F, Vester-Glowinski P, Rasmussen BS, et al. Mesenchymal stem cell therapy of acute thermal burns: A systematic review of the effect on inflammation and wound healing. Burns. 2021;47(2):270–294. Tamama K, Kerpedjieva SS. Acceleration of Wound Healing by Multiple Growth Factors and Cytokines Secreted from Multipotential Stromal Cells/Mesenchymal Stem Cells. Adv Wound Care (New Rochelle). 2012;1(4):177–182. Yu B, Zhang X, Li X. Exosomes derived from mesenchymal stem cells. Int J Mol Sci. 2014;15(3):4142–57. Mendt M, Kamerkar S, Sugimoto H, et al. Generation and testing of clinical-grade exosomes for pancreatic cancer. JCI Insight. 2018;3(8):e99263. Marote A, Teixeira FG, Mendes-Pinheiro B, et al. MSCs-Derived Exosomes: Cell-Secreted Nanovesicles with Regenerative Potential. Front Pharmacol. 2016;7:231. Zhang J, Guan J, Niu X, et al. Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med. 2015;13:49. Zhang B, Wang M, Gong A, et al. HucMSCs-Exosome Mediated-Wnt4 Signaling Is Required for Cutaneous Wound Healing. Stem Cells. 2015;33(7):2158–68. Li X, Liu L, Yang J, et al. Exosome Derived From Human Umbilical Cord Mesenchymal Stem Cell Mediates MiR-181c Attenuating Burn-induced Excessive Inflammation. EBioMedicine. 2016;8:72–82. Hu L, Wang J, Zhou X, et al. Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts. Sci Rep. 2016;6:32993. Ma T, Fu B, Yang X, et al. Adipose mesenchymal stem cell-derived exosomes promote cell proliferation, migration, and inhibit cell apoptosis via Wnt/β-catenin signaling in cutaneous wound healing. J Cell Biochem. 2019;120(6):10847–10854. Xiu C, Zheng H, Jiang M, Li J, Zhou Y, Mu L, Liu W. MSCs-Derived miR-150-5p-Expressing Exosomes Promote Skin Wound Healing by Activating PI3K/AKT Pathway through PTEN. Int J Stem Cells. 2022;15(4):359–371. He X, Dong Z, Cao Y, et al. MSCs-Derived Exosome Promotes M2 Polarization and Enhances Cutaneous Wound Healing. Stem Cells Int. 2019;2019:7132708. Dalirfardouei R, Jamialahmadi K, Jafarian AH, et al. Promising effects of exosomes isolated from menstrual blood-derived mesenchymal stem cell on wound-healing process in diabetic mouse model. J Tissue Eng Regen Med. 2019;13(4):555–568. Li B, Luan S, Chen J, et al. The MSCs-Derived Exosomal lncRNA H19 Promotes Wound Healing in Diabetic Foot Ulcers by Upregulating PTEN via MicroRNA-152-3p. Mol Ther Nucleic Acids. 2020;19:814–826. Hui Y Z, Xi G C, Ming K, et al. Preparation of chitosan-based thermosensitive hydrogels for drug delivery[J]. Journal of Applied Polymer Science, 2009, 112(3):1509–1515. 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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-4564135","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":319447285,"identity":"be1df36c-7e88-45bc-bb5c-1520c8ef8070","order_by":0,"name":"Lei Xu","email":"","orcid":"","institution":"General Hospital of the Western Theater Command","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Xu","suffix":""},{"id":319447287,"identity":"876f99ba-62c5-48c3-9b19-666c43841522","order_by":1,"name":"Dan Liu","email":"","orcid":"","institution":"General Hospital of the Western Theater Command","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Liu","suffix":""},{"id":319447288,"identity":"f327b5ed-90a3-43b9-b3f3-f3db8fb7d8d0","order_by":2,"name":"Hai Long Yun","email":"","orcid":"","institution":"General Hospital of the Western Theater Command","correspondingAuthor":false,"prefix":"","firstName":"Hai","middleName":"Long","lastName":"Yun","suffix":""},{"id":319447289,"identity":"863d0386-f1a8-4db7-8a57-5017e662ad4b","order_by":3,"name":"Wei Zhang","email":"","orcid":"","institution":"General Hospital of the Western Theater Command","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zhang","suffix":""},{"id":319447290,"identity":"67dc4539-d68b-40a2-a324-1fe3ddc7a29e","order_by":4,"name":"Li Ren","email":"","orcid":"","institution":"General Hospital of the Western Theater Command","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Ren","suffix":""},{"id":319447291,"identity":"b8b3dae6-1564-4b9d-928b-c50ff37de089","order_by":5,"name":"Wen Wen Li","email":"","orcid":"","institution":"General Hospital of the Western Theater Command","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"Wen","lastName":"Li","suffix":""},{"id":319447292,"identity":"f63c192f-c877-4c73-b92e-6124121bd04c","order_by":6,"name":"Chuan Han","email":"data:image/png;base64,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","orcid":"","institution":"General Hospital of the Western Theater Command","correspondingAuthor":true,"prefix":"","firstName":"Chuan","middleName":"","lastName":"Han","suffix":""}],"badges":[],"createdAt":"2024-06-11 12:36:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4564135/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4564135/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59608004,"identity":"94dc8ca5-fe51-4409-8745-4cd44ffc5e00","added_by":"auto","created_at":"2024-07-03 19:09:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1396169,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of ASCs-Exos. a. A large number of vesicle-like aggregations with double-concave bilayer membrane structures of 30-150 nm in size were observed under 15k and 40k electron microscopy. b. WB assay confirmed the expression of exosome markers CD9, CD63, and CD81.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4564135/v1/c1ba9a2f465cd9829604ec96.jpg"},{"id":59608007,"identity":"9c14bab8-f136-4f03-8636-cb1cb1950e94","added_by":"auto","created_at":"2024-07-03 19:09:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1788852,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of CS-αβ-GP thermosensitive hydrogel crosslinked with ASCs-Exos on the healing rate of deep burn wounds in rats. a. Photographs of wound healing in the CS+ASCs-Exos group, ASCs-Exos group, CS group, and control group immediately after wound formation and at 2, 4, 6, 8, 10, 12, and 14 days after treatment. b. Clustered bar chart showing the wound healing rates (%) in the CS+ASCs-Exos group, ASCs-Exos group, CS group, and control group immediately after wound formation and at 2, 4, 6, 8, 10, 12, and 14 days after treatment.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4564135/v1/9a8370e1fd9814677bb65e35.jpg"},{"id":59608006,"identity":"6f4f0d6b-31ee-4b7b-909c-1816efcaf405","added_by":"auto","created_at":"2024-07-03 19:09:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5604520,"visible":true,"origin":"","legend":"\u003cp\u003eThe inflammatory response of the wound treated with CS-αβ-GP thermosensitive hydrogel cross-linked with ASCs-Exos. a. (HE) CS+ASCs-Exos: smooth muscle cells and fibroblasts are visible under the microscope in normal tissues, with a small number of capillaries. Loose fibroblasts and collagen fibers are visible under the microscope in the wound edge tissue, with many small arteries and veins, and scattered lymphocytes. Myofibroblasts, fibroblasts, newly formed capillaries, and infiltrating lymphocytes are visible under the microscope in the wound tissue. ASCs-Exos: smooth muscle cells and fibroblasts are visible under the microscope in normal tissues, with a small number of capillaries and glands. Loose fibroblasts and collagen fibers are visible under the microscope in the wound edge tissue, with many small arteries and veins, and scattered lymphocytes. Myofibroblasts, fibroblasts, glandular cells, and infiltrating lymphocytes are visible under the microscope in the wound tissue. CS: smooth muscle cells and fibroblasts are visible under the microscope in normal tissues, with a small number of capillaries. Small granulation tissue is visible under the microscope in the wound edge tissue. Smooth muscle cells and fibroblasts are visible under the microscope in the wound tissue, with a small number of capillaries and glands. Ctrl: smooth muscle cells and fibroblasts are visible under the microscope in normal tissues, with a small number of capillaries and glands. Glandular cells are visible under the microscope in the wound edge tissue. Many fibroblasts and randomly distributed capillaries are visible under the microscope in the wound tissue. b. (Masson) CS+ASCs-Exos: a large number of muscle fibers are visible under the microscope in normal tissues, with a small amount of collagen fibers. More muscle fibers and a small amount of increased collagen fibers are visible under the microscope in the wound edge tissue. The number and distribution of collagen fibers and muscle fibers in the wound tissue are consistent with those in the wound edge tissue. ASCs-Exos: muscle fibers and a large number of collagen fibers are visible under the microscope in normal tissues. The number and distribution of collagen fibers and muscle fibers in the wound edge tissue and wound tissue are consistent with those in the CS+ASCs-Exos group. CS: muscle fibers and a large number of collagen fibers are visible under the microscope in normal tissues. Muscle fibers and a large number of collagen fibers are visible under the microscope in the wound edge tissue. Abundant collagen fibers and a small number of muscle fibers are visible under the microscope in the wound tissue. Ctrl: muscle fibers and a large number of collagen fibers are visible under the microscope in normal tissues. A large number of muscle fibers and a small amount of collagen fibers are visible under the microscope in the wound edge tissue and wound tissue. c. (IHC) The expression of IL-10 and TGF-β in the wound tissue of the CS+ASCs-Exos group is slightly higher than in the other groups, indicating stronger anti-inflammatory effects than the other groups. The expression of TNF-α, IL-1α, IL-6, and IL-10 in the wound tissue of the CS+ASCs-Exos group is lower than in the other groups, indicating lower proinflammatory effects than the other groups. The expression of EGF in the CS+ASCs-Exos group is also higher than in the other groups, indicating that it can accelerate wound healing.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4564135/v1/7e38e263b5220e6fd8f1ee84.jpg"},{"id":59608005,"identity":"4fa0f840-f9a6-42cf-b678-d95e7844c97c","added_by":"auto","created_at":"2024-07-03 19:09:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":206957,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of CS-αβ-GP thermosensitive hydrogel cross-linked with ASCs-Exos on wound macrophages. The expressions of IL-1α and CD86 in the CS+ASCs-Exos group and the ASCs-Exos group were lower than those in the control group, and the expression in the CS+ASCs-Exos group was even lower. The expressions of CCL22 and CD163 in the CS+ASCs-Exos group and the ASCs-Exos group were higher than those in the control group, and the expression in the CS+ASCs-Exos group was even higher.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4564135/v1/19b2c201fbc5614949cb528a.jpg"},{"id":62406215,"identity":"cf76af24-624c-4ace-a033-90b8872087c0","added_by":"auto","created_at":"2024-08-13 21:01:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9602942,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4564135/v1/0a93a139-29a1-4d7a-8d40-a4f38af70bb3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effect of Adipose-derived stem cells exosomes cross-linked Chitosan-αβ-glycerophosphate thermosensitive hydrogel on deep burn wounds","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eHuman skin is the largest organ of the body, whose main function is to provide appropriate protection and prevent external factors and pathogens from invading the body. As wars and natural disasters gradually increase, burns have become a huge challenge affecting skin integrity. Although there have been many studies related to burns, there are still great challenges in the management and treatment of burn wounds. In recent years, with the development of regenerative medicine and tissue engineering, there are endless means for chronic refractory wounds, among which MSCs-Exos(Mesenchymal stem cell exosomes)are expected to become the best measure to solve this problem.\u003c/p\u003e \u003cp\u003eA large amount of research evidence indicates that MSCs-Exos can promote the healing of burn wounds, and compared with MSCs, they have significant advantages in terms of storage stability, ease of collection, and safety [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, current studies exploring the use of MSCs-Exos for wound repair mainly adopt subcutaneous injection and intravenous injection as the main administration methods, which face the problems of burst release and rapid clearance, making it difficult to play their due role in the complex stages of wound repair. Currently, research is underway to load MSCs-Exos onto patches, injectable microcarriers, or hydrogels to maintain the function of Exos(Exosomes) at the wound site and improve efficiency and safety. Multiple studies have shown that various artificial injectable hydrogels can not only promote the sustained release of Exos in the body but also increase the local retention of Exos [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These hydrogel dressings include CS (Chitosan), alginate, urea pyridine ketone, polypeptide-based FHE hydrogel, etc., among which CS exhibits high antibacterial activity against fungi, bacteria, algae, and viruses [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and creates a moist healing environment for the wound. Therefore, this study was designed to investigate the effect of CS-αβ-GP (Chitosan-αβ-glycerophosphate) thermosensitive hydrogel cross-linked with ASCs-Exos (Adipose-derived stem cells exosomes) on deep burn wounds and verify whether CS-αβ-GP thermosensitive hydrogel is the best carrier for loading MSCs-Exos to promote the healing of deep burn wounds.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Isolation and Culture of ASCs-Exos\u003c/h2\u003e\n \u003cp\u003eThe SD rat ASCs (product code: RASMD-01001) were purchased from OriCell. The 1 mL of cells were aspirated with a sterile pipette into a 5 mL sterile EP tube, diluted with 9 mL of basic medium, and centrifuged at 1000 rpm for 5 minutes to allow the cells to settle. The supernatant was discarded. Four milliliters of freshly prepared medium containing 10% serum were added, gently mixed, and the cells were transferred to a 25T culture flask. The culture flask was placed flat and mixed in a figure-eight pattern before being placed in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for culture. After 24 hours of culture, the cells were observed under a microscope to ensure adherence, and the medium was carefully replaced for continued culture until the cells reached 90%-100% confluence, at which point the medium was discarded. One milliliter of PBS was added to rinse the cells, repeated three times, and the PBS was discarded. Four milliliters of complete medium were then added. Half of the detached cells were transferred to a new culture flask, and each flask was supplemented with complete medium to 4 mL. The flasks were carefully sealed, placed flat, mixed in a figure-eight pattern, and placed in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for culture. Once the cells reached confluence, they were passaged using the same method. Cells with good vitality and approximately 70% density were selected for cryopreservation. They were transferred to a sterile EP tube and centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The resulting pellet contained the ASCs-Exos. Freshly prepared cell cryopreservation solution was added, with 2 mL per 25T cell culture, and the solution was divided into two cryopreservation tubes, aiming for a cell density of 5x10\u003csup\u003e6\u003c/sup\u003e-1x10\u003csup\u003e7\u003c/sup\u003e cells per mL. The tubes were placed in a -80\u0026deg;C freezer overnight, and the next day, the cryopreservation tubes were quickly transferred to liquid nitrogen for long-term storage.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Identification of ASCs-Exos\u003c/h2\u003e\n \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.1 Electron microscopy observation\u003c/h2\u003e\n \u003cp\u003eCells were taken out from the incubator. The concentration of Exos was adjusted to 0.5g/L, resuspended with PBS, and fixed with 2.5% glutaraldehyde for 10 minutes. 20\u0026ndash;30\u0026micro;L of the fixed Exos suspension was taken and dropped onto a carbon-coated copper grid and allowed to stand at room temperature for 1 minute. It was then dried under an infrared lamp for 5 minutes. 1% phosphotungstic acid was added dropwise onto the copper grid and stained at room temperature for 5 minutes. The phosphotungstic acid solution was then absorbed and dried again under an infrared lamp for 10 minutes. Finally, the copper grid was placed under a transmission electron microscope for observation and imaging.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.2 WB (western blot) Detection of Protein Expression Level\u003c/h2\u003e\n \u003cp\u003eCells were taken out from the incubator, and the cell culture medium was discarded. The cells were washed with PBS once. The PBS was discarded, and cell lysate and PMSF were added with a ratio of 100:1. The mixture was placed on ice at 4℃. The cells were fully lysed by pipetting, and the cell lysate samples were transferred to a centrifuge tube and further lysed on ice for 10\u0026thinsp;~\u0026thinsp;15 minutes. Then, the samples were centrifuged at 12,000 rpm for 10 minutes at 4℃. The supernatant was transferred to a new centrifuge tube, and loading buffer was added. The samples were heated at 100℃ for 20 minutes in a thermostat. After that, the samples were centrifuged at 12,000 rpm for 1 minute at 4℃ and stored at -20℃ for future use. After gel preparation and sample loading, electrophoresis and immunostaining were performed.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.3 The preparation of thermosensitive hydrogel cross-linked ASCs-Exos with CS-\u0026alpha;\u0026beta;-GP\u003c/h2\u003e\n \u003cp\u003eAdd 2g of CS with 75% deacetylation degree to 100ml of acetic acid/sodium acetate buffer solution with a given pH value of 4.6, and stir until it is completely dissolved. Then, filter out impurities with a 100-mesh cell sieve, sterilize it in a high-pressure sterilizer, and cool it in an ice bath for 20 minutes. Prepare a 50% W/V (weight/volume) \u0026alpha;\u0026beta;-GP aqueous solution in distilled water, sterilize it in a high-pressure sterilizer, and add it dropwise to the CS solution in the ice bath while stirring according to the ratio of CS/\u0026alpha;\u0026beta;-GP at 8.8/1.2. Cool the final mixed CS-\u0026alpha;\u0026beta;-GP solution in the ice bath for 20 minutes, and store it at 4℃ for later use. Mix Exos with the CS-\u0026alpha;\u0026beta;-GP hydrogel to obtain a working solution of CS-Exos with a mass concentration of 100g/L of Exos. After incubating it at 37℃ for 30 minutes, the Exos cross-linked CS-\u0026alpha;\u0026beta;-GP thermo-sensitive hydrogel is formed.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Animal and Ethical Statement\u003c/h2\u003e\n \u003cp\u003eForty 8-week-old healthy SPF-grade male Sprague Dawley (SD) rats, with body weights ranging from 200 to 220 grams, were purchased from the Experimental Animal Center of Western Theater General Hospital. After purchase, the rats were conventionally kept for one week of adaptation under conditions of (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2) ℃, 40%-60% humidity, and a 12-hour light/dark cycle, with free access to food and water. The animal study was approved by the Ethics Committee of the General Hospital of the Western Theater Command (Ethics Approval Number: XBZQZYY2021-046).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Establishment of deep second-degree burn model in rats\u003c/h2\u003e\n \u003cp\u003eThe rats were anesthetized by intravenous injection of thiopental sodium (40mg/kg) in the tail. After shaving the hair on the back, the skin on the back was scalded with 80℃ water for 8 seconds, with a wound diameter of 16mm. Then, the wound was covered with sterile gauze soaked in physiological saline for 6 minutes. Debridement was not performed on all rats. The rats with deep burns were divided into the following groups:\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003ea. CS-ASCs-Exos group: the CS-\u0026alpha;\u0026beta;-GP thermosensitive hydrogel cross-linked with Exos containing 200\u0026micro;g Exos was locally applied to cover the full-thickness skin wound, and a transparent dressing was applied over it. The wound dressing was replaced every 2 days.\u003c/p\u003e\n \u003c/span\u003e\u003cspan\u003e\n \u003cp\u003eb. CS group: the hydrogel with the same volume as the CS-\u0026alpha;\u0026beta;-GP thermosensitive hydrogel cross-linked with Exos containing 200\u0026micro;g Exos was locally applied to cover the full-thickness skin wound, and a transparent dressing was applied over it. The wound dressing was replaced every 2 days.\u003c/p\u003e\n \u003c/span\u003e\u003cspan\u003e\n \u003cp\u003ec. ASCs-Exos group: 200\u0026micro;g Exos was dissolved in 200\u0026micro;l PBS and locally dispersed onto the full-thickness skin wound using a pipette, and a transparent dressing was applied over it. The wound dressing was replaced every 2 days.\u003c/p\u003e\n \u003c/span\u003e\u003cspan\u003e\n \u003cp\u003ed. Control group: only a transparent dressing was locally applied to cover the wound, and it was replaced every 2 days without further treatment.\u003c/p\u003e\n \u003c/span\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Assessment of wound healing\u003c/h2\u003e\n \u003cp\u003eImmediately after the formation of wounds and at 2, 4, 6, 8, 10, 12, and 14 days after treatment, the wound healing of rats in each group was observed and recorded. Image J software was used to analyze and calculate the wound healing rate at each time point. The wound healing rate = (1 - unhealed wound area / original wound area) \u0026times; 100%.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Pathological assessment of wound healing\u003c/h2\u003e\n \u003cp\u003eAfter 14 days of treatment, full-thickness skin tissues of the wounds and wound edges in the four groups were excised, respectively. Normal full-thickness skin tissues were also excised from the corresponding positions on the dorsal wounds of the rats. After fixation with 40 g/L paraformaldehyde for 24 h, dehydration, paraffin embedding, and slicing (4 \u0026micro;m) were performed successively. Finally, HE(hematoxylin-eosin) staining was used for staining. Masson staining was used to observe the deposition of collagen fibers in the wound tissues.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Immunohistochemical assessment of wound\u003c/h2\u003e\n \u003cp\u003eImmunohistochemical methods were used to detect the expression of TNF-\u0026alpha;, TGF-\u0026beta;, IL-1\u0026alpha;, IL-6, IL-10, and EGF (Epidermal growth factor) in newly formed wound tissue. TNF-\u0026alpha;, IL-1\u0026alpha;, and IL-6 were used to observe the proinflammatory status of the wound in rats, while IL-10 and TGF-\u0026beta; were used to observe the anti-inflammatory status of the wound in rats. EGF was used to observe the wound healing status.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 qPCR (Real-time quantitative polymerase chain reaction) Detection of Gene Expression\u003c/h2\u003e\n \u003cp\u003eThe deep second-degree burn model of rats was constructed according to the modeling method in 2.5. After 14 days of treatment according to the experimental grouping, RNA was extracted from the wound surface and qPCR was used to detect the mRNA expression levels of IL-1\u0026alpha;, CD86, CCL22, and CD163. IL-1\u0026alpha; and CD86 are markers of M1 macrophages, while CCL22 and CD163 are markers of M2 macrophages.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterization of ASCs-Exos\u003c/h2\u003e \u003cp\u003eTo ensure the reliability of subsequent experiments, we first characterized ASCs-Exos. The commercially purchased ASCs cells were cultured, and then 100ml of supernatant from the culture medium was taken for exosome extraction. Transmission electron microscopy was used for observation and identification. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, a large number of vesicle-like enrichments with double-concave bilayer membrane structures were observed visually under the electron microscope, with sizes ranging from 30 to 150 nm. Then, 100ml of supernatant from the culture medium of ASCs cells was taken for exosome extraction, and WB was used to detect the expression of typical exosome markers CD9, CD63, and CD81. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, after culturing and passage of ASCs cells, CD9, CD63, and CD81 protein expressions were detected in the extracts. These results indicate that the exosome isolation was successful and subsequent experiments can be carried out.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 Accelerating wound healing in deep burn injuries in rats through cross-linking ASCs-Exos with CS-αβ-GP thermosensitive hydrogel\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo demonstrate that CS-αβ-GP thermosensitive hydrogel is an effective carrier for transporting Exos, we cross-linked ASCs-Exos to CS-αβ-GP thermosensitive hydrogel and applied it to a deep second-degree burn injury model in rats to verify its effect. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, on days 0, 2, 4, 6, 8, 10, 12, and 14 after wound formation and treatment, the wound healing rate in the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group was significantly higher from day 4 onward compared to the CS group, ASCs-Exos group, and control group. The final healing rate on day 14 was also higher than the other three groups, indicating that CS\u0026thinsp;+\u0026thinsp;ASCs-Exos can accelerate wound healing and is superior to both the CS hydrogel group and the ASCs-Exos group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Reducing Inflammation of Wound Surface after Cross-Linking ASCs-Exos with CS-αβ-GP thermosensitive hydrogel\u003c/h2\u003e \u003cp\u003eTo verify the effect of Exos cross-linked with CS-αβ-GP thermosensitive hydrogel on the wound surface, full-thickness skin tissues of the wound and wound margin from the three groups were resected after 14 days of treatment, and normal full-thickness skin tissues were resected from the corresponding positions on the back of the rats for HE staining and Masson staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, HE staining revealed that the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group had a small number of inflammatory cells, a small amount of blood vessels, and muscle and collagen fibers distributed at intervals consistent with normal tissues at the wound margin on day 14, indicating that ASCs-Exos cross-linked with CS-αβ-GP thermosensitive hydrogel can reduce the inflammatory response of the wound surface. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Masson staining showed that the collagen and muscle fibers in the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group were distributed at intervals in the wound and wound margin on day 14, consistent with normal tissues. In other groups, the staining of collagen fibers was stronger than that in the experimental group, suggesting that the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group produced less collagen fibers and had a better outcome of scar repair. Then, IHC (immunohistochemical) staining was performed on the skin tissues to detect the expression of TNF-α, IL-1α, IL-6, IL-10, TGF-β, and EGF. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. The expressions of IL-10, TGF-β, and EGF in the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group were slightly higher than those in other groups, while the expressions of TNF-α, IL-1α, IL-6, and IL-10 were lower than those in other groups, indicating that CS\u0026thinsp;+\u0026thinsp;ASCs-Exos can increase the anti-inflammatory effect, reduce inflammatory factors, and promote wound healing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 After crosslinking with CS-αβ-GP thermosensitive hydrogel, ASCs-Exos promote the transformation of M1 macrophages into M2 macrophages.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further verify the effect of Exos crosslinked with CS-αβ-GP thermosensitive hydrogel on macrophages, qPCR was used to detect the mRNA expression levels of M1 macrophage markers IL-1α, CD86, and M2 macrophage markers CCL22, CD163. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the expressions of IL-1α and CD86 in the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group were lower than those in the control group, while the expressions of CCL22 and CD163 were higher than those in the control group, indicating that CS\u0026thinsp;+\u0026thinsp;ASCs-Exos can promote the transformation of M1 macrophages to M2 macrophages, and the transformation effect is stronger compared with the ASCs-Exos group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eMSCs refer to multipotent progenitor cells that can differentiate into osteoblasts, chondrocytes, and adipocytes under standard in vitro differentiation conditions. MSCs are divided into BM-MSCs (Bone-marrow derived stem cells), ASCs (Adipose-derived stem cells), UC-MSCs (Umbilical cord-derived stem cells), iPSC-MSCs (human induced pluripotent stem cell-derived MSCs) and BD-MSCs (Burn tissue-derived mesenchymal stem cells) according to their sources. Among them, ASCs can be obtained from autologous or allogeneic subcutaneous adipose tissues of the abdomen, thighs, and arms through liposuction and separation. Almost 5,000 ASCs can be extracted from 1 gram of adipose tissue, which is easy to obtain and has received increasing attention. Studies have shown that MSCs are recruited to the damaged tissue sites during tissue injury and activated under the close interaction of the inflammatory environment and the immune system [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In response to the inflammatory environment, MSCs secrete some cytokines and growth factors and change the composition of local cytokines, which is considered beneficial to wound healing and tissue regeneration [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additionally, MSCs have been proven to promote wound healing by increasing angiogenesis, inhibiting inflammation, and promoting fibroblast migration and collagen production through paracrine mechanisms [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. All these characteristics are considered crucial for rapid wound healing and are expected to control wound infection, prevent the progression of burn wounds, accelerate skin healing, restore skin barrier function, and reduce scar formation in the treatment of burns.\u003c/p\u003e \u003cp\u003eHowever, MSCs have issues such as tumorigenicity, immune rejection, infection transmission, and complex material storage, which limit their applications. Compared with MSCs, MSCs-Exos have the following advantages: (1) Long-term storage stability: MSCs-Exos are about one millionth of the size of MSCs, have lower complexity, stable structure, are easy to produce and store, and are not affected by storage at -20\u0026deg;C for one week. Their activity can be maintained even after long-term storage at -80\u0026deg;C [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. (2) Easy to collect: Various types of MSCs can secrete Exos, and each type can produce 1000 to 10000 of them. Exosomes can be extracted from the culture medium through methods such as ultracentrifugation, and specialized cell lines can also be used to produce Exos on a large scale. Compared with MSCs, the production of MSCs-Exos is simpler, cheaper, and less time-consuming [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. (3) Safety: Cell-based therapies using MSCs have issues such as cell survival, regenerative capacity, immune rejection, and tumor differentiation. As a cell-free therapy, Exos can avoid these problems. Due to their low membrane-bound protein content, the possibility of immune rejection is very low even after allogeneic administration. Additionally, Exos do not proliferate, thus eliminating the possibility of tumor formation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, MSCs-Exos have better stability, economy, and safety than MSCs in clinical applications.\u003c/p\u003e \u003cp\u003eResearch on skin wound healing has shown that MSCs-Exos can accelerate skin healing and reduce excessive scar formation. Zhang et al. injected human iPSC-MSCs-Exos subcutaneously around the wound site of a rat model and evaluated the efficacy of human iPSC-MSCs-Exos by measuring wound closure area, histology, and immunofluorescence. The results showed that human iPSC-MSCs-Exos implanted into the wound could accelerate epithelial reformation, reduce scar width, and promote collagen maturation. In addition, it not only promoted the formation of new blood vessels but also accelerated their maturation at the wound site. Furthermore, iPSC-MSCs-Exos stimulated the proliferation and migration of human dermal fibroblasts and human umbilical vein endothelial cells in a dose-dependent manner in vitro and promoted the secretion of type I and III collagen and elastin [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Another study used human UC-MSCs-Exos injected subcutaneously into a rat model with deep second-degree burn wounds on the skin, which showed a significant acceleration of epithelial reformation and increased expression of CK19 (Cytokeratin 19), PCNA (Proliferating cell nuclear antigen), and collagen I (Compared to collagen III) in vivo. In vivo studies confirmed that MSCs-Exos-mediated activation of Wnt/β-catenin could promote wound re-epithelialization and cell proliferation. Knockout of Wnt4 in MSCs-Exos reduced the therapeutic effect in vivo [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Li et al. established a rat model of third-degree burns and intravenously injected human UC-MSCs-Exos. They found that the endogenous miR-181c in MSCs-Exos could inhibit the TLR4 signaling pathway, thereby reducing LPS (Lipopolysaccharide)-mediated inflammation. Administration of human UC-MSCs-Exos overexpressing miR-181c reversed the upregulation of inflammatory factors such as TNF-α and IL-1β and the downregulation of the anti-inflammatory factor IL-10 caused by burns [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Hu et al. studied the role of ASCs-Exos in skin wound healing. The results showed that both local injection and intravenous injection of ASCs-Exos could be taken up and internalized by fibroblasts, stimulating cell migration, proliferation, and collagen synthesis in a dose-dependent manner. It also increased the gene expression of N-cadherin, cyclin-1, PCNA, and collagen I and III. In the early stages of wound healing, systemic administration of ASCs-Exos increased the production of collagen I and III, while in the later stages, it might inhibit collagen expression to reduce scar formation. This suggests that ASCs-Exos can promote skin wound healing by optimizing the characteristics of fibroblasts [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Ma et al. exposed HaCaT cells (Human immortalized keratinocytes) to H2O2 to establish a skin lesion model and then used human ASCs-Exos. They found that ASCs-Exos could promote the proliferation and migration of HaCaT cells and inhibit their apoptosis. In addition, the enhancement of β-catenin at the protein level confirmed the activation of the Wnt/β-catenin signaling pathway, suggesting that ASCs-Exos may promote skin wound healing through Wnt/β-catenin signaling [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Macrophages are divided into M1 and M2 subtypes based on their activation status. M1 macrophages release proinflammatory factors such as TNF-α, IL-1α, IL-1β, IL-6, CXCL9 (C-X-C motif chemokine ligand 9), and CXCL10, while M2 macrophages secrete anti-inflammatory mediators, including IL-10, TGF-β, CCL1 (C-C motif chemokine ligand 1), CCL17, CCL18, and CCL22 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. He et al. intravenously injected human BM-MSCs-Exos into mice with full-thickness skin excision and showed that miR-223 in MSCs-Exos induced macrophage polarization to the M2 subtype by targeting pknox1, thereby accelerating skin wound healing. This demonstrates that miRNAs in MSCs-Exos can be used to promote skin wound healing [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Dalirfardouei et al. established full-thickness excision wounds on the backs of diabetic mice induced by streptozotocin to simulate diabetic foot ulcers. Then, they used menstrual blood-derived MSCs-Exos for subcutaneous injection and observed that MSCs-Exos enhanced neovascularization by upregulating vascular endothelial growth factor A, inhibited inflammation by inducing M1 macrophage polarization to M2, accelerated re-epithelialization, and reduced scar formation by lowering the Col1 (Collagen type 1):Col3 ratio. This suggests that menstrual blood-derived MSCs-Exos can improve the non-healing of diabetic foot ulcers [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Another study using a mouse model of diabetic foot ulcer showed that intravenous injection of BM-MSCs-Exos overexpressing lncRNA H19 prevented the apoptosis and inflammation of fibroblasts by weakening the PTEN (Phosphatase with tensin homology) inhibition mediated by miR-152-3p, thus promoting wound healing in mice with diabetic foot ulcer [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough a large amount of research evidence suggests that MSCs-Exos can promote the healing of burn wounds, the current main administration methods are subcutaneous injection and intravenous injection, which face the problems of burst release and rapid clearance, which are not conducive to wound healing. Multiple studies have shown that various injectable artificial hydrogels can not only promote the sustained release of Exos in vivo, but also increase the local retention of Exos [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Hydrogel dressings include CS, alginate, ureido pyrimidinone, peptide-based FHE hydrogel, etc. Among them, CS also shows high antibacterial activity against fungi, bacteria, algae, and viruses, and creates a wet healing environment for the wound [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. CS-αβ-GP hydrogel is a flowable viscous liquid at temperatures below 37\u0026deg;C, which turns into a non-flowable gel when heated to 37\u0026deg;C. CS-αβ-GP thermosensitive hydrogel with a pH of 4.6, an ionic strength of 0.15 mol/L, and a CS/αβ-GP ratio of 8.8/1.2 is stable at 4\u0026deg;C for at least 3 months, so it is hopeful to become the best carrier for loading MSCs-Exos [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur findings reveal that the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group exhibited a marked enhancement in wound healing rates starting from day 4, ultimately surpassing the healing rates observed in the CS group, ASCs-Exos group, and control group on day 14. This suggests that CS\u0026thinsp;+\u0026thinsp;ASCs-Exos can accelerate wound healing and is superior to the other groups. HE staining revealed that in the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group on day 14, there were fewer inflammatory cells at the wound margin, with a small number of blood vessels, and the distribution of muscle fibers and collagen fibers was consistent with normal tissue. This suggests that the combination of ASCs-Exos and CS-αβ-GP thermosensitive hydrogel can reduce the inflammatory response at the wound site. Masson staining showed that in the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group on day 14, the wound and wound margin exhibited a distribution of collagen fibers and muscle fibers consistent with normal tissue. In contrast, the staining of collagen fibers in the other groups was stronger than in the experimental group, indicating that the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group produced less collagen fiber and had a better outcome of scar repair. IHC staining revealed that the expressions of IL-10, TGF-β, and EGF in the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group were slightly higher than in the other groups, while the expressions of TNF-α, IL-6, IL-1α, and IL-10 were lower. This suggests that CS\u0026thinsp;+\u0026thinsp;ASCs-Exos can increase anti-inflammatory effects, reduce inflammatory factors, and promote wound healing. qPCR analysis showed that the expressions of IL-1α and CD86 in the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group were lower than in the control group, while the expressions of CCL22 and CD163 were higher. This indicates that CS\u0026thinsp;+\u0026thinsp;ASCs-Exos can promote the transformation of M1 macrophages to M2 macrophages, and this effect is stronger compared to the ASCs-Exos group.\u003c/p\u003e \u003cp\u003eIn summary, our study has demonstrated that ASCs-Exos cross-linked CS-αβ-GP thermosensitive hydrogel has anti-inflammatory effects, promotes wound healing, and facilitates the transition of M1 macrophages to M2 macrophages. Moreover, its effects are stronger compared to ASCs-Exos alone. However, there are still some limitations in our research. First, it is unclear how ASCs-Exos cross-linked CS-αβ-GP thermosensitive hydrogel regulates signaling pathways. Second, the molecular substances enriched after ASCs-Exos cross-linked CS-αβ-GP thermosensitive hydrogel remain to be studied. Third, in the current study, we only verified the effects of ASCs-Exos cross-linked CS-αβ-GP thermosensitive hydrogel on deep burn wounds in animal models. Nevertheless, our results provide a new method of administration for the clinical application of MSCs-Exos. Further clinical studies are needed to validate our findings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclarations\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eEthical Approval and consent to participate\u003c/strong\u003e \u003cp\u003eThe animal study was approved by the Ethics Committee of the General Hospital of the Western Theater Command (Ethics Approval Number: XBZQZYY2021-046).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication:\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThere are no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the incubation program of the General Hospital of the Western Theater Command (Nos. 2021-XZYG-C29 and 2021-XZYG-C45). The funders had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHan, Xu, Liu, Zhang, Ren and Li wrote the main manuscript text. Xu and Yun prepared Figure 1-4. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Dr Xian Hui Li from the Department of Burns in General Hospital of the Western Theater Command, and Dr Hao Yao from Department of hematology in General Hospital of the Western Theater Command for their contributions to the study.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYu B, Zhang X, Li X. Exosomes derived from mesenchymal stem cells. Int J Mol Sci. 2014;15(3):4142\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMendt M, Kamerkar S, Sugimoto H, et al. Generation and testing of clinical-grade exosomes for pancreatic cancer. JCI Insight. 2018;3(8):e99263.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarote A, Teixeira FG, Mendes-Pinheiro B, Salgado AJ. MSCs-Derived Exosomes: Cell-Secreted Nanovesicles with Regenerative Potential. Front Pharmacol. 2016;7:231.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi Q, Qian Z, Liu D, et al. GMSCs-Derived Exosomes Combined with a Chitosan/Silk Hydrogel Sponge Accelerates Wound Healing in a Diabetic Rat Skin Defect Model. Front Physiol. 2017;8:904.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShafei S, Khanmohammadi M, Heidari R, et al. Exosome loaded alginate hydrogel promotes tissue regeneration in full-thickness skin wounds: An in vivo study. J Biomed Mater Res A. 2020;108(3):545\u0026ndash;556.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMol EA, Lei Z, Roefs MT, et al. Injectable Supramolecular Ureidopyrimidinone Hydrogels Provide Sustained Release of Extracellular Vesicle Therapeutics. Adv Healthc Mater. 2019;8(20):e1900847.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoy R C, Morais S T B, Assis O B G. Evaluation of the antimicrobial activity of chitosan and its quaternized derivative on E. coli and S. aureus growth[J]. Revista Brasileira de Farmacognosia, 2016, 26: 122\u0026ndash;127.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRangatchew F, Vester-Glowinski P, Rasmussen BS, et al. Mesenchymal stem cell therapy of acute thermal burns: A systematic review of the effect on inflammation and wound healing. Burns. 2021;47(2):270\u0026ndash;294.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamama K, Kerpedjieva SS. Acceleration of Wound Healing by Multiple Growth Factors and Cytokines Secreted from Multipotential Stromal Cells/Mesenchymal Stem Cells. Adv Wound Care (New Rochelle). 2012;1(4):177\u0026ndash;182.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu B, Zhang X, Li X. Exosomes derived from mesenchymal stem cells. Int J Mol Sci. 2014;15(3):4142\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMendt M, Kamerkar S, Sugimoto H, et al. Generation and testing of clinical-grade exosomes for pancreatic cancer. JCI Insight. 2018;3(8):e99263.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarote A, Teixeira FG, Mendes-Pinheiro B, et al. MSCs-Derived Exosomes: Cell-Secreted Nanovesicles with Regenerative Potential. Front Pharmacol. 2016;7:231.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Guan J, Niu X, et al. Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med. 2015;13:49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang B, Wang M, Gong A, et al. HucMSCs-Exosome Mediated-Wnt4 Signaling Is Required for Cutaneous Wound Healing. Stem Cells. 2015;33(7):2158\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, Liu L, Yang J, et al. Exosome Derived From Human Umbilical Cord Mesenchymal Stem Cell Mediates MiR-181c Attenuating Burn-induced Excessive Inflammation. EBioMedicine. 2016;8:72\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu L, Wang J, Zhou X, et al. Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts. Sci Rep. 2016;6:32993.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa T, Fu B, Yang X, et al. Adipose mesenchymal stem cell-derived exosomes promote cell proliferation, migration, and inhibit cell apoptosis via Wnt/β-catenin signaling in cutaneous wound healing. J Cell Biochem. 2019;120(6):10847\u0026ndash;10854.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiu C, Zheng H, Jiang M, Li J, Zhou Y, Mu L, Liu W. MSCs-Derived miR-150-5p-Expressing Exosomes Promote Skin Wound Healing by Activating PI3K/AKT Pathway through PTEN. Int J Stem Cells. 2022;15(4):359\u0026ndash;371.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe X, Dong Z, Cao Y, et al. MSCs-Derived Exosome Promotes M2 Polarization and Enhances Cutaneous Wound Healing. Stem Cells Int. 2019;2019:7132708.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDalirfardouei R, Jamialahmadi K, Jafarian AH, et al. Promising effects of exosomes isolated from menstrual blood-derived mesenchymal stem cell on wound-healing process in diabetic mouse model. J Tissue Eng Regen Med. 2019;13(4):555\u0026ndash;568.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi B, Luan S, Chen J, et al. The MSCs-Derived Exosomal lncRNA H19 Promotes Wound Healing in Diabetic Foot Ulcers by Upregulating PTEN via MicroRNA-152-3p. Mol Ther Nucleic Acids. 2020;19:814\u0026ndash;826.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHui Y Z, Xi G C, Ming K, et al. Preparation of chitosan-based thermosensitive hydrogels for drug delivery[J]. Journal of Applied Polymer Science, 2009, 112(3):1509\u0026ndash;1515.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-4564135/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4564135/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThis study aimed to explore the effects on controlling infection and promoting wound healing in deep burn injuries by crosslinking ASCs-Exos with CS-αβ-GP thermosensitive hydrogel.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eRats with established deep burn injury models were divided into four groups: CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group, ASCs-Exos group, CS group, and control group. The wound healing rates were analyzed and calculated using Image J software immediately after wound formation and on days 2, 4, 6, 8, 10, 12, and 14 after treatment. Fourteen days after treatment, skin tissues from the wound area, wound margin, and normal full-thickness skin were excised from each group for HE staining and Masson staining. Subsequently, IHC staining was performed on the newly formed wound tissues to detect the expression of TNF-α, IL-6, IL-1α, IL-10, TGF-β, and EGF. Finally, RNA was extracted from the wound tissues, and qPCR was used to detect the mRNA expression levels of IL-1α, CD86, CCL22, and CD163.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe wound healing rate in the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group was higher than that in the other groups. HE staining revealed that the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group had fewer inflammatory cells, a small number of blood vessels, and muscle fibers and collagen fibers distributed alternately in the wound edge at 14 days, which was consistent with normal tissue. Masson staining showed that the wound and wound edge in the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group at 14 days displayed alternating distributions of collagen fibers and muscle fibers, which was consistent with normal tissue. However, the staining of collagen fibers in the other groups was stronger than that in the experimental group. IHC staining showed that the expressions of IL-10, TGF-β, and EGF in the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group were slightly higher than those in the other groups, while the expressions of TNF-α, IL-6, IL-1α, and IL-10 were lower than those in the other groups. qPCR detection revealed that the expressions of IL-1α and CD86 in the CS\u0026thinsp;+\u0026thinsp;ASCs-Exos group were lower than those in the control group, while the expressions of CCL22 and CD163 were higher than those in the control group.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur research has demonstrated that ASCs-Exos crosslinked with CS-αβ-GP thermosensitive hydrogel exhibits anti-inflammatory properties, promotes wound healing, and enhances the transformation of M1 macrophages into M2 macrophages, with stronger effects compared to ASCs-Exos alone. This provides a new administration method for the clinical application of MSCs-Exos.\u003c/p\u003e","manuscriptTitle":"The effect of Adipose-derived stem cells exosomes cross-linked Chitosan-αβ-glycerophosphate thermosensitive hydrogel on deep burn wounds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 19:09:05","doi":"10.21203/rs.3.rs-4564135/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":"8ceaec38-7ddb-4161-bc5b-4187719270ad","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-13T20:53:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-03 19:09:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4564135","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4564135","identity":"rs-4564135","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}