Effect of hucMSC-derived exosomes on the pressure ulcers in mice

Effect of hucMSC-derived exosomes on the pressure ulcers in mice | 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 Effect of hucMSC-derived exosomes on the pressure ulcers in mice Fei Yan, Junzheng Yang, Meihua Gong, Guangheng Li, Li Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7005414/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 Background : Pressure ulcers (PUs) are a result of sustained compression of the local skin, leading to impaired blood circulation and subsequent nutritional deficiencies in the skin and subcutaneous tissues. This condition can result in damage, ulceration, and even necrosis. It serious threat to the patient's life and health, with the potential to result in fatal consequences. (MSCs) have been shown to secrete exosomes, termed MSC-exos, which range in size from 40 to 160 nm, and have been found to contain DNA, lipids, and proteins, and have been implicated in the treatment of various diseases. However, the effect of hucMSC-exos on the PU and the underlying mechanisms remain not to be fully elucidated. Methods : The present study investigates the effects of exosome isolation from hucMSCs on the mouse PU model. Following the administration of hucMSC-exos, the healing of the wound and the histopathological changes in the mice were evaluated by HE staining and Masson staining; the collagen mRNA levels in the tissues were analyzed by quantitative real-time polymerase chain reaction (qRT–PCR); the expressions of HMGB1, α-SMA, and CD34 were examined by western blotting and immunohistochemistry to investigate the potential mechanisms by which the therapeutic effect of hucMSC-exos on PU is achieved. Results : The results of HE staining and Masson staining demonstrated that hucMSC-exos could alleviate the histopathological symptoms of PU. qRT–PCR results demonstrated that hucMSC-exos decreased the levels of collagen I, collagen III, TGF-β, and HGMB1 in PU mice, the results of western blotting demonstrated that the expressions of HMGB1, SMA, and CD34 could also be decreased by the administration of hucMSC-exos. Conclusions: The present study demonstrated that hucMSC-exos alleviated the symptoms of PU by regulating HGMB1 and α-SMA. which demonstrated that the therapeutic potential of hucMSC-exos in PU. exosomes pressure ulcer hucMSCs HMGB1 α-SMA Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Pressure ulcers (PU) are local tissue injuries caused by long-term pressure on the skin and/or underlying tissues in bony protrusion body areas, blood circulation disorders, continuous ischemia/hypoxia, and malnutrition. It is well established that pressure or shear forces can compromise the structural integrity of various anatomical tissues, including the epidermis, dermis, adipose tissue, muscle and bone. This damage can potentially result in the onset of ischemia–reperfusion (I-R) injury, which, in turn, can trigger the development of chronic refractory skin ulcers [ 1 – 2 ]. In the context of recurrent I-R cycles have been demonstrated to be more detrimental to skin tissue than simple long-term ischemia, and the reperfusion period impacts tissue damage [ 3 ]. During repeated I-R events, the infiltration of leukocyte, oxidative stress, infection, and inflammation that ensues collectively generate an imbalance between pro- and anti-inflammatory factors. results in a reduction in tissue perfusion and subsequent necrosis [ 4 ]. Repetitive I-R events have been demonstrated to unfavorable conditions for the delivery of growth factors that are necessary for the repair and homeostasis of damaged tissues [ 5 ]. PU has been observed to affect elderly populations more significantly, the treatment of PU remains challenging, underscoring the necessity for the development of novel therapeutic interventions. Recent studies have demonstrated the significance of paracrine signaling from tissue stem cells in promoting wound healing and the regeneration of damaged tissue [ 6 ]. Exosomes are defined as secreted vesicles measuring between 50 and 150 nanometres in diameter. They contain proteins, lipids and nucleic acids, and are capable of delivering the paracrine signals between cells. This suggests that they represent a therapeutic opportunity for tissue repair and regeneration. Exosomes derived from human umbilical cord mesenchymal stem cells (hucMSC-exos) have been identified as a a source and delivery system for a significant number of cytokines and growth factors. These have the capacity to influence the tissue response to injury, thereby promoting wound healing [ 7 ]. Exosome-based delivery systems as therapeutic molecules is of significant interest due to their high biodegradability, low toxicity, immunogenicity, and the capacity to protect their internalised cargo [ 8 – 10 ]. The present study sought to evaluate the effect of hucMSC-exos on PU and to explore the potential mechanism involved in a mouse skin wound model. The objective was to understand the therapeutic effect of hucMSC-exos on PU and the underlying mechanism. 2. Materials and Methods 2.1 Animal experiments and experiment design Fifteen 6-week-old male BALB/c specific pathogen-free (SPF) mice were purchased from the Guangdong Medical Laboratory Animal Center. All experimental and animal care procedures were approved by the Animal Care and Use Committee of Shenzhen People’s Hospital. All protocols were approved by the Animal Ethics Committee of Shenzhen People’s Hospital (LL-LK-201928) and conducted in full compliance with the Guide of the Care and Use of Laboratory Animals of China. The mice were randomly divided into three groups (normal group, model group, and treatment group). The mice in model group and treatment group were underwent three I-R cycles for modeling. Following acclimatizing the environment for 1 week, the procedure for pressure ulcer wound model was conducted. Firstly, the mice were anesthetized using isoflurane inhalation (1.8%), following which the dorsal back was shaved and cleaned with 75% alcohol. The dorsal back was then subsequently pinched between two circular magnets (Dongguan JinKun Magnet products, CO) that were 10mm in diameter, 5 mm thick, and had a strength of 4000 G. The full-thickness skin was pulled up and placed between a pair of magnetic disks. The dorsal skin was exposed to a 12-hour cycle of magnetic pressure and subsequent rest (referred to as an I-R cycle). A total of 3 rounds of I-R were performed. The compressed area was left uncovered for the rest of the procedure after the PU wound induction protocol had been performed. The mice in different group were evaluated at 1, 7 and 14 days. The wounds were subjected to daily evaluated by histopathological analysis; with the reduction in wound size being calculated using the following equation: wound-size reduction (%)= (AO-At)/AO×100, where AO is the initial wound area, and At is the wound area at each day. The fluorescence was measured at 12-hour intervals by bioluminescence imaging at day 1, day 2, and day 3 following the injection of the DIR-labeled exosomes into the wounds. 2.2 HE staining and Masson staining On the 14th day, the cutaneous wound bed tissues were meticulously dissected, then fixed in 4% phosphate-buffered formalin (pH 7.4). Following this, the tissues were embedded in paraffin, sectioned at a thickness of 5 µm, and mounted on glass slides for further analysis. The sections were subjected to hematoxylin and eosin (H&E) staining and Masson staining in accordance with standard procedures, and were then observed under a light microscope (Olympus BX53 L220US). 2.3 RT-qPCR RT–qPCR was used to examined the mRNA levels of Type I collagen, Type III collagen, and transforming growth factor-β (TGF-β) in PU mice. The expression levels of the three aforementioned genes were normalized to the housekeeping gene GAPDH. The primer sequences utilized in this study are delineated below: Type I collagen: (forward-5′-TGACCGATGGATTCCCGTTC-3′, reverse-5′- GCAGGGCCTTCTTGAGGTTG-3′); Type III collagen: (forward-5′- AAGCACTGGTGGACAGATTC-3′, reverse-5′-CGGCTGGAAAGAAGTCTGAG-3′); GAPDH: (forward-5′-AGCTTGTCATC AACGGGAAG-3′, reverse-5′- TTTGATGTTAGTGGGGTCTCG-3′); and TGF-β: (forward-5'-ACAACCCACACCTGATCCTC-3', reverse-5'-GTTCGTGGACCC ATTTCCAG-3'); HMGB1: (forward-5'-CTTCCTCATCCTCTTCATCC-3', reverse-5'-GCCCTATGAGAA GAA AGC TG-3'). 2.4 Immunohistochemistry staining The evaluation of inflammation and angiogenesis was conducted by utilizing biotinylated antibodies against alpha-smooth muscle actin (α-SMA) (Abcam), the endothelial marker CD34 (Abcam) and HMGB1 (R&D Systems) for the labeling of PU wound sections. To assess the number of vessels, the entire area of the wound on the slide was observed under a microscope, the number of vessels was counted in each of the four quadrants of the wound, the number of vessels was counted in each of the four quadrants of the wound, and the average was calculated. The evaluation of staining was conducted by four independent researchers, who were unaware of the experimental conditions. 2.5 Cell Culture hucMSCs were purchased from Cyagen (stock number: HUXUC-01001 batch number: 160117I31). MSCs were cultivated in an incubator at 37°C with 5% CO 2 , and were passage every 3 days. The medium was then replaced with TM-ACF PLUS Medium Human (MesenCult) supplemented with a cell growth supplement (MesenCult TM-ACF PLUS 500X Supplement). MSCs were passaged at a ratio of 1:2 in T175 flasks, and the culture medium was collected for exosome purification when the cell confluence reached 90%. 2.6 Isolation and identification of exosomes hucMSCs were purchased from Cyagen (stock number: HUXUC-01001 batch number: 160117I31). The cells were then cultured in an incubator at 37°C with 5% CO 2 , and were passaged every 3 days. Subsequent to five passages of hucMSCs, the culture medium was replaced with TM-ACF PLUS Medium Human (MesenCult) supplemented with a cell growth supplement (MesenCult TM-ACF PLUS 500X Supplement). In order to isolate exosomes, the medium of hucMSCs was collected and subjected to centrifugation at 400×g for 10min, 2000×g for 10min, and 10,000×g for 30min to remove the cell fragments and protein aggregates. The collected supernatant was then subjected to further centrifugation at 100,000×g for 120min in order to obtain the exosomes. The procedure for purifying the exosome involved the removal of the upper layer (the 'supernatant') and the subsequent washing of the exosome pellet with ice-cold Phosphate Buffered Saline (PBS). The washing step was followed by centrifugation at 100,000×g for a duration of 120 minutes. The exosome pellets were then stored in a -80°C freezer. In order to identify the exosome samples, the surface markers of CD9, CD63, and CD81 were analyzed by western blotting; the protein levels of the exosome samples were measured using a BCA protein assay kit (Pierce Protein Biology; Thermo Fisher Scientific Life Sciences); and the shape and size of the exosome samples were analyzed by transmission electron microscopy (TEM) (Tecnai G2 Spirit Biotwin). 2.7 DIR-labeled exosomes HucMSC-exos were labeled with DIR (1,1ʹ-dioctadecyl-3,3,3ʹ,3ʹ-tetramethylindotricarbocyanine iodide; Invitrogen, Carlsbad, CA, USA), a near-infrared fluorescent membrane dye, and then precipitated using ExoQuick-TCTM (System Biosciences, USA). Following the process of centrifugation at 10,000×g for 10min, the labeled exosomes were resuspended in PBS and stored at − 4°C for subsequent use. 2.8 Western blotting The collected proteins from tissues or cells were firstly lysed by RIPA buffer. The protein concentrations were subsequently measured using a BCA protein assay kit. For the purpose of western blot analysis, primary antibodies directed against HMGB1 (purchased from Sigma–Aldrich) and α-SMA were employed, with GAPDH serving as an internal control. The blots were subsequently developed using ECL reagent. 2.9 Statistical analysis The descriptive data were expressed as the mean ± SD. GraphPad Prism version 6 (GraphPad software) was used to analyze the data. Student’s t test or analysis of variance was used to compared the differences between two groups. and p < 0.05 were considered statistically significant. 3. Results 3.1 The characteristics of hucMSC-exos and the uptake of hucMSC-exos in PU mice The results of TEM and nanoparticle tracking analysis (NTA, ZetaView PMX 110, Particle Metrix, Germany) demonstrated that the morphology of hucMSC-exos was characterized by a circular single-layer membrane structure, with a diameter ranging from 30 to 150nm (Fig. 1 A and Fig. 1 C). The results of the western blotting analysis demonstrated that hucMSC-exos exhibited high levels of expression of the markers CD9, CD63, and CD81 but not cainexin (Fig. 1 B). Furthermore, to evaluate whether exosome uptake and localization in the wound after treatment, PU mice were injected with DIR-labeled hucMSC-exos. The results demonstrated that the signal was detectable immediately in the mice body following hucMSC-exos administration and remained so for a period of 48h, indicating that hucMSC-exos could be taken up by the PU mice and may exert a continuous therapeutic effect (Fig. 1 D). 3.2 hucMSC-exos promoted the cutaneous wound healing in PU mice The experimental design of the effect of the hucMSC-exos on PU mice were illustrated in Fig. 2 A. The representative images of the modeling method and the symptoms of PU mice were shown in Fig. 2 B. The results demonstrated that the wound healing of PU mice was improved following the hucMSC-exos treatment in comparison with the model group at day 1, day 3, day 7, and day 14 (Fig. 2 C); and an accelerated wound healing rate was also observed at day 1, day 3, day 7, and day 14 following hucMSC-exos treatment (Fig. 2 D). These data demonstrated that hucMSC-exos could promote the wound healing of PU mice in vivo. 3.3 Effects of hucMSC-exos on histopathological changes of PU mice In order to evaluate the effect of hucMSC-exos on the histopathological changes in PU mice, hematoxylin and eosin (HE) staining and Masson staining were conducted. The results demonstrated that hucMSC-exos decreased the wound scar areas and regular epidermal and dermal structures in PU mice compared with the PBS group (Fig. 3 A); and densely packed with collagen fibers in the dermal layer, and arranged in parallel in the epidermis were observed following hucMSC-exos treatment (Fig. 3 B). RT-qPCR results demonstrated that hucMSC-exos decreased the expressions of α-SMA, CD34, collagen I, and collagen III in PU mice (Fig. 3 D); and further immunochemistry results demonstrated that the expression of α-SMA and CD34 were also decreased in the PU mice following the administration of hucMSC-exos (Fig. 3 C). These data demonstrated that the beneficial effects of hucMSC-exos treatment on wound healing and skin tissue regeneration in PU mice. 3.4 The underlying mechanisms of hucMSC-exos treatment on PU mice The results of immunohistochemical analysis demonstrated that expression of HMGB1 was high in epidermal tissues of the PU mice, and the expression of HMGB1 was significantly decreased following the administration of hucMSC-exos (Fig. 6C). In addition, the results of the western blotting analysis demonstrated a decline in the expressions of HMGB1 and α-SMA following the treatment with hucMSC-exos (Fig. 4 A and Fig. 4 B). These results demonstrated that the HMGB1/α-SMA axis regulated the therapeutic effect of hucMSC-exos in PU mice. 4. Discussion PUs are a type of skin lesion that has been identified as the primary cause of I-R, which is associated with an anti-inflammatory phase characterized by severe defects in cell-mediated immunity [ 16 ]. It is evident that a multitude of many cellular elements and intricate molecular interactions are involved in this inflammatory reaction. hucMSC-exos, which contain various bioactive molecules, including mRNAs and microRNAs, have been shown to facilitate intracellular communication [ 17 ]. Moreover, these exosomes have been demonstrated to exert significant paracrine effects on wound repair, promoting cell migration and metastasis while concomitantly attenuating the inflammation associated with wound healing [ 18 ]. In this article, we isolated the exosomes from hucMSCs firstly, further confirmed that hucMSCs-exos could be uptake by PU mice, and the data demonstrated that the administration of hucMSCs-exos has the capacity to impede cutaneous wound healing and to ameliorate the histopathological changes in PU mice. These results indicated that hucMSC-exos had the good drug safety and therapeutic effects on PU. In normal skin, CD34 is primarily located in endothelial cells, dermal dendritic cells, and perifollicular cells. It has been demonstrated that CD34 + cells have a significant role in the process of wound healing, they have the capacity to enhance the epithelial regeneration, mechanical support, intercellular communication, and immune regulation [ 19 , 20 ]. is a protein that plays a pivotal role in maintaining the structural integrity and elasticity of various bodily tissues, including the skin, bones, and tendons. It is also involved in crucial processes such as organ and tissue development, cell migration, proliferation, and differentiation, wound healing, and tissue remodeling. Among the various types of collagen, collagen I and collagen III are particularly prominent, accounting for the highest proportion [ 21 , 22 ]. α-SMA is commonly found in fibroblasts, smooth muscle cells, and muscle cells. It is significantly upregulated shortly after the inflammatory phase of skin trauma and plays a crucial role in the formation of the extracellular matrix [ 23 ]. It has been demonstrated that the function of MSC-derived exosome varies at different stages of the wound-healing process. In the initial phase of wound healing, the levels of type III collagen, type I collagen, and CD34 increased following the administration of MSC-derived exosome therapy. During the remodelling phase, the expression levels of type III collagen, type I collagen, and α-SMA decreased [ 24 , 25 ]. In this article, the data demonstrated that the expressions of type III collagen, type I collagen, and α-SMA were decreased after the treatment of hucMSC-exos for for a period of 14 days. These data demonstrated that hucMSC-exos exhibited effective tissue repair function and reduced scar formation in PU mice. HMGB1 is a chromatin-bound nuclear protein that can also act as a cytokine under stress conditions or during cell death. has been identified as a pivotal factor in the induction of sterile immunity, with its release by ischemic cells being a crucial element in the activation of the inflammatory pathway [ 26 , 27 ]. The release of HMGB1 from the cell is a significant event in the injury process, as it plays a crucial role in the inflammatory response, akin to that of a cytokine. Furthermore, HMGB1 has been demonstrated to induce fibrosis and scar formation, while the inhibition of HMGB1 has been shown to impede fibroblast migration and collagen synthesis, thereby significantly reducing scarring [ 28 ]. Our data demonstrated that the high expression of HMGB1 was observed in the dermis layer of PU mice, and that the expression of HMGB1 was significantly decreased by hucMSC-exos. This was further confirmed by RT-qPCR and western blotting. These data demonstrated HMGB1 may may have a role in regulating the treatment of PU with hucMSC-exos. 5. Conclusions In this study, we confirmed that hucMSC-exos had the therapeutic effect on the promotion of tissue re-epithelialization and vascularization of PU by regulating HMGB1 and α-SMA, which may provide a promising strategy for the treatment of PU. Declarations Authors’ contributions FY, MHG, JZY and LY contributed to data analysis and interpretation, data collection, literature review, and drafted the manuscript. FRL and LY contributed to the conception and design of the study. FY, and MHG contributed to the MSC production and characterization. FY, MHG, JZY, FRL and LY participated in the data collection and interpretation. All authors critically reviewed and approved the final manuscript. Acknowledgments All authors have made substantial contributions to the conception or design of the work and to revising and drafting the manuscript. Funding This work was supported by the Translational Medicine Collaborative Innovation Center and Plastic and Aesthetic Department in Shenzhen People’s Hospital. This study was supported financially by the Hospital young and middle-aged research backbone training project (1033). Ethics approval and consent to participate This work was approved by the Ethics Committee of the Medical Ethics Committee of Shenzhen People’s Hospital. The medical ethical number: LL-LK-201928 Availability of data and materials All materials are available from the corresponding author. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Sun J, Wang ZJ, Wang XR (2018) Suppression of LRRC19 promotes cutaneous wound healing in pressure ulcers in mice. Organogenesis 14(1):13–24. 10.1080/15476278.2018.1436924 Sundin BM, Hussein MA, Glasofer S et al (2000) The role of allopurinol and deferoxamine in preventing pressure ulcers in pigs. 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J Leukoc Biol 93(6):865–873. 10.1189/jlb.1212662 Yang H, Hreggvidsdottir HS, Palmblad K, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA (2010) A critical cysteine is required for HMGB1 binding to Toll-like receptor 4 and activation of macrophage cytokine release., 107 (26): 11942–11947. 10.1073/pnas.1003893107 Tang ST, Wang F, Shao M et al (2017) MicroRNA-126 suppresses inflammation in endothelial cells under hyperglycemic condition by targeting HMGB1.VASCULAR PHARMACOLOGY. 88:48–55. 10.1016/j.vph.2016.12.002 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7005414","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":492272483,"identity":"809c7d50-888d-4749-b8b2-940fbae86448","order_by":0,"name":"Fei Yan","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Yan","suffix":""},{"id":492272484,"identity":"8a0d9eb7-c4f1-4143-89f6-bd41bc8daa15","order_by":1,"name":"Junzheng Yang","email":"data:image/png;base64,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","orcid":"","institution":"Jinan University","correspondingAuthor":true,"prefix":"","firstName":"Junzheng","middleName":"","lastName":"Yang","suffix":""},{"id":492272487,"identity":"4b524e4b-b1f9-47b2-957a-13fe9faf9737","order_by":2,"name":"Meihua Gong","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Meihua","middleName":"","lastName":"Gong","suffix":""},{"id":492272489,"identity":"e40bdfe2-7213-4806-a1ff-25a0d55086e7","order_by":3,"name":"Guangheng Li","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Guangheng","middleName":"","lastName":"Li","suffix":""},{"id":492272490,"identity":"bb0456fd-650b-4552-9448-099ac99883c0","order_by":4,"name":"Li Yu","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2025-06-30 02:08:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7005414/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7005414/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88002016,"identity":"6c2524f5-48b3-4505-9e45-2f8359e23b7c","added_by":"auto","created_at":"2025-07-31 10:25:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2347176,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe morphology of hucMSC-exos and the \u0026nbsp;uptake of hucMSC-exos in PU mice.\u003c/strong\u003e (A) TEM analysis the morphology of hucMSC-Exos isolated from hucMSCs (scale bar=0.5μm); (B) Western blotting detected the expressions of CD9, CD63, CD81, and \u0026nbsp;cainexin in hucMSC-exos; (C) NTA analyzed the size distribution of hucMSC-exos; (D) The\u003cstrong\u003e \u003c/strong\u003euptake experiment demonstrated the hucMSC-exos could be kept in the PU mice for 48h.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7005414/v1/46b4089c31ea047d0c6f0a0e.png"},{"id":88002021,"identity":"574ef4d4-f952-4488-ba0b-8c0b4a7d0791","added_by":"auto","created_at":"2025-07-31 10:25:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1483706,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehucMSC-exos alleviated the cutaneous wounds in PU mice.\u003c/strong\u003e (A) The experimental flow chart. (B) The representative images of PU mice model were induced by a pair of magnet disks. (C) The representative images of the effect of hucMSC-exos on wound mice at day 1, day 3, day 7, and day 14. (d) The statistical analysis the wound closure following hucMSC-exos treatment. *P \u0026lt; 0.05, **P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7005414/v1/f1bd5cb09ace0b9041458eff.png"},{"id":88002019,"identity":"fced9572-b0bc-4714-a9bb-634a77888d9f","added_by":"auto","created_at":"2025-07-31 10:25:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5067299,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehucMSC-exos alleviated the histopathological changes in PU mice. \u003c/strong\u003e(A) HE staining observed the effect of hucMSC-exos on wound re-epithelialization; the double-headed arrows indicated the edges of the scar; (B) HE staining and Masson staining observed the\u003cstrong\u003e \u003c/strong\u003ehistopathological changes in PU mice (scale bar =100μm); (C) Immunohistochemical analysis of the expressions of α-SMA and CD34 in PU mice following 14-day treatment of hucMSC-exos (scale bar=100μm); (D) RT-qPCR analyzed the expressions of α-SMA, CD34, collagen I, and collagen III in PU mice. *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7005414/v1/c55669ed40db47d2ac1c19ff.png"},{"id":88003750,"identity":"e82dd75f-bdd3-4b61-99b3-cfcbfcd22646","added_by":"auto","created_at":"2025-07-31 10:33:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1476685,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical and western blotting analysis the underlying mechanisms of hucMSC-exos treatment on PU mice\u003c/strong\u003e. (A) Western blot analysis of the expression of HMGB1 and α-SMA after hucMSC-exos treatment. (B) The quantitative analysis of the data obtained from western blotting. *P\u0026lt;0.05, **P\u0026lt;0.01. (C) Immunohistochemical analysis of the expression of HMGB1 in PU mice (scale bar = 100μm or 50μm).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7005414/v1/0e7cdf58637809ea0498a4c8.png"},{"id":88377540,"identity":"7870fe17-af3f-492c-aadd-97e277290b11","added_by":"auto","created_at":"2025-08-05 22:16:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11422680,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7005414/v1/96fefecb-145b-47fd-8d86-c288a10442fb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of hucMSC-derived exosomes on the pressure ulcers in mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePressure ulcers (PU) are local tissue injuries caused by long-term pressure on the skin and/or underlying tissues in bony protrusion body areas, blood circulation disorders, continuous ischemia/hypoxia, and malnutrition. It is well established that pressure or shear forces can compromise the structural integrity of various anatomical tissues, including the epidermis, dermis, adipose tissue, muscle and bone. This damage can potentially result in the onset of ischemia\u0026ndash;reperfusion (I-R) injury, which, in turn, can trigger the development of chronic refractory skin ulcers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In the context of recurrent I-R cycles have been demonstrated to be more detrimental to skin tissue than simple long-term ischemia, and the reperfusion period impacts tissue damage [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. During repeated I-R events, the infiltration of leukocyte, oxidative stress, infection, and inflammation that ensues collectively generate an imbalance between pro- and anti-inflammatory factors. results in a reduction in tissue perfusion and subsequent necrosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Repetitive I-R events have been demonstrated to unfavorable conditions for the delivery of growth factors that are necessary for the repair and homeostasis of damaged tissues [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. PU has been observed to affect elderly populations more significantly, the treatment of PU remains challenging, underscoring the necessity for the development of novel therapeutic interventions.\u003c/p\u003e\u003cp\u003eRecent studies have demonstrated the significance of paracrine signaling from tissue stem cells in promoting wound healing and the regeneration of damaged tissue [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Exosomes are defined as secreted vesicles measuring between 50 and 150 nanometres in diameter. They contain proteins, lipids and nucleic acids, and are capable of delivering the paracrine signals between cells. This suggests that they represent a therapeutic opportunity for tissue repair and regeneration. Exosomes derived from human umbilical cord mesenchymal stem cells (hucMSC-exos) have been identified as a a source and delivery system for a significant number of cytokines and growth factors. These have the capacity to influence the tissue response to injury, thereby promoting wound healing [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Exosome-based delivery systems as therapeutic molecules is of significant interest due to their high biodegradability, low toxicity, immunogenicity, and the capacity to protect their internalised cargo [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The present study sought to evaluate the effect of hucMSC-exos on PU and to explore the potential mechanism involved in a mouse skin wound model. The objective was to understand the therapeutic effect of hucMSC-exos on PU and the underlying mechanism.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animal experiments and experiment design\u003c/h2\u003e\u003cp\u003eFifteen 6-week-old male BALB/c specific pathogen-free (SPF) mice were purchased from the Guangdong Medical Laboratory Animal Center. All experimental and animal care procedures were approved by the Animal Care and Use Committee of Shenzhen People\u0026rsquo;s Hospital. All protocols were approved by the Animal Ethics Committee of Shenzhen People\u0026rsquo;s Hospital (LL-LK-201928) and conducted in full compliance with the Guide of the Care and Use of Laboratory Animals of China.\u003c/p\u003e\u003cp\u003eThe mice were randomly divided into three groups (normal group, model group, and treatment group). The mice in model group and treatment group were underwent three I-R cycles for modeling. Following acclimatizing the environment for 1 week, the procedure for pressure ulcer wound model was conducted. Firstly, the mice were anesthetized using isoflurane inhalation (1.8%), following which the dorsal back was shaved and cleaned with 75% alcohol. The dorsal back was then subsequently pinched between two circular magnets (Dongguan JinKun Magnet products, CO) that were 10mm in diameter, 5 mm thick, and had a strength of 4000 G. The full-thickness skin was pulled up and placed between a pair of magnetic disks. The dorsal skin was exposed to a 12-hour cycle of magnetic pressure and subsequent rest (referred to as an I-R cycle). A total of 3 rounds of I-R were performed. The compressed area was left uncovered for the rest of the procedure after the PU wound induction protocol had been performed. The mice in different group were evaluated at 1, 7 and 14 days. The wounds were subjected to daily evaluated by histopathological analysis; with the reduction in wound size being calculated using the following equation: wound-size reduction (%)= (AO-At)/AO\u0026times;100, where AO is the initial wound area, and At is the wound area at each day. The fluorescence was measured at 12-hour intervals by bioluminescence imaging at day 1, day 2, and day 3 following the injection of the DIR-labeled exosomes into the wounds.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 HE staining and Masson staining\u003c/h2\u003e\u003cp\u003eOn the 14th day, the cutaneous wound bed tissues were meticulously dissected, then fixed in 4% phosphate-buffered formalin (pH 7.4). Following this, the tissues were embedded in paraffin, sectioned at a thickness of 5 \u0026micro;m, and mounted on glass slides for further analysis. The sections were subjected to hematoxylin and eosin (H\u0026amp;E) staining and Masson staining in accordance with standard procedures, and were then observed under a light microscope (Olympus BX53 L220US).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 RT-qPCR\u003c/h2\u003e\u003cp\u003eRT\u0026ndash;qPCR was used to examined the mRNA levels of Type I collagen, Type III collagen, and transforming growth factor-β (TGF-β) in PU mice. The expression levels of the three aforementioned genes were normalized to the housekeeping gene GAPDH. The primer sequences utilized in this study are delineated below: Type I collagen: (forward-5\u0026prime;-TGACCGATGGATTCCCGTTC-3\u0026prime;, reverse-5\u0026prime;- GCAGGGCCTTCTTGAGGTTG-3\u0026prime;); Type III collagen: (forward-5\u0026prime;- AAGCACTGGTGGACAGATTC-3\u0026prime;, reverse-5\u0026prime;-CGGCTGGAAAGAAGTCTGAG-3\u0026prime;); GAPDH: (forward-5\u0026prime;-AGCTTGTCATC AACGGGAAG-3\u0026prime;, reverse-5\u0026prime;- TTTGATGTTAGTGGGGTCTCG-3\u0026prime;); and TGF-β: (forward-5'-ACAACCCACACCTGATCCTC-3', reverse-5'-GTTCGTGGACCC ATTTCCAG-3'); HMGB1: (forward-5'-CTTCCTCATCCTCTTCATCC-3', reverse-5'-GCCCTATGAGAA GAA AGC TG-3').\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Immunohistochemistry staining\u003c/h2\u003e\u003cp\u003eThe evaluation of inflammation and angiogenesis was conducted by utilizing biotinylated antibodies against alpha-smooth muscle actin (α-SMA) (Abcam), the endothelial marker CD34 (Abcam) and HMGB1 (R\u0026amp;D Systems) for the labeling of PU wound sections. To assess the number of vessels, the entire area of the wound on the slide was observed under a microscope, the number of vessels was counted in each of the four quadrants of the wound, the number of vessels was counted in each of the four quadrants of the wound, and the average was calculated. The evaluation of staining was conducted by four independent researchers, who were unaware of the experimental conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Cell Culture\u003c/h2\u003e\u003cp\u003ehucMSCs were purchased from Cyagen (stock number: HUXUC-01001 batch number: 160117I31). MSCs were cultivated in an incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, and were passage every 3 days. The medium was then replaced with TM-ACF PLUS Medium Human (MesenCult) supplemented with a cell growth supplement (MesenCult TM-ACF PLUS 500X Supplement). MSCs were passaged at a ratio of 1:2 in T175 flasks, and the culture medium was collected for exosome purification when the cell confluence reached 90%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Isolation and identification of exosomes\u003c/h2\u003e\u003cp\u003ehucMSCs were purchased from Cyagen (stock number: HUXUC-01001 batch number: 160117I31). The cells were then cultured in an incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, and were passaged every 3 days. Subsequent to five passages of hucMSCs, the culture medium was replaced with TM-ACF PLUS Medium Human (MesenCult) supplemented with a cell growth supplement (MesenCult TM-ACF PLUS 500X Supplement). In order to isolate exosomes, the medium of hucMSCs was collected and subjected to centrifugation at 400\u0026times;g for 10min, 2000\u0026times;g for 10min, and 10,000\u0026times;g for 30min to remove the cell fragments and protein aggregates. The collected supernatant was then subjected to further centrifugation at 100,000\u0026times;g for 120min in order to obtain the exosomes. The procedure for purifying the exosome involved the removal of the upper layer (the 'supernatant') and the subsequent washing of the exosome pellet with ice-cold Phosphate Buffered Saline (PBS). The washing step was followed by centrifugation at 100,000\u0026times;g for a duration of 120 minutes. The exosome pellets were then stored in a -80\u0026deg;C freezer. In order to identify the exosome samples, the surface markers of CD9, CD63, and CD81 were analyzed by western blotting; the protein levels of the exosome samples were measured using a BCA protein assay kit (Pierce Protein Biology; Thermo Fisher Scientific Life Sciences); and the shape and size of the exosome samples were analyzed by transmission electron microscopy (TEM) (Tecnai G2 Spirit Biotwin).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 DIR-labeled exosomes\u003c/h2\u003e\u003cp\u003eHucMSC-exos were labeled with DIR (1,1ʹ-dioctadecyl-3,3,3ʹ,3ʹ-tetramethylindotricarbocyanine iodide; Invitrogen, Carlsbad, CA, USA), a near-infrared fluorescent membrane dye, and then precipitated using ExoQuick-TCTM (System Biosciences, USA). Following the process of centrifugation at 10,000\u0026times;g for 10min, the labeled exosomes were resuspended in PBS and stored at \u0026minus;\u0026thinsp;4\u0026deg;C for subsequent use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Western blotting\u003c/h2\u003e\u003cp\u003eThe collected proteins from tissues or cells were firstly lysed by RIPA buffer. The protein concentrations were subsequently measured using a BCA protein assay kit. For the purpose of western blot analysis, primary antibodies directed against HMGB1 (purchased from Sigma\u0026ndash;Aldrich) and α-SMA were employed, with GAPDH serving as an internal control. The blots were subsequently developed using ECL reagent.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e\u003cp\u003eThe descriptive data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. GraphPad Prism version 6 (GraphPad software) was used to analyze the data. Student\u0026rsquo;s t test or analysis of variance was used to compared the differences between two groups. and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1 The characteristics of hucMSC-exos and the uptake of hucMSC-exos in PU mice\u003c/h2\u003e\u003cp\u003eThe results of TEM and nanoparticle tracking analysis (NTA, ZetaView PMX 110, Particle Metrix, Germany) demonstrated that the morphology of hucMSC-exos was characterized by a circular single-layer membrane structure, with a diameter ranging from 30 to 150nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The results of the western blotting analysis demonstrated that hucMSC-exos exhibited high levels of expression of the markers CD9, CD63, and CD81 but not cainexin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, to evaluate whether exosome uptake and localization in the wound after treatment, PU mice were injected with DIR-labeled hucMSC-exos. The results demonstrated that the signal was detectable immediately in the mice body following hucMSC-exos administration and remained so for a period of 48h, indicating that hucMSC-exos could be taken up by the PU mice and may exert a continuous therapeutic effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2 hucMSC-exos promoted the cutaneous wound healing in PU mice\u003c/h2\u003e\u003cp\u003eThe experimental design of the effect of the hucMSC-exos on PU mice were illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. The representative images of the modeling method and the symptoms of PU mice were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. The results demonstrated that the wound healing of PU mice was improved following the hucMSC-exos treatment in comparison with the model group at day 1, day 3, day 7, and day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC); and an accelerated wound healing rate was also observed at day 1, day 3, day 7, and day 14 following hucMSC-exos treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These data demonstrated that hucMSC-exos could promote the wound healing of PU mice in vivo.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Effects of hucMSC-exos on histopathological changes of PU mice\u003c/h2\u003e\u003cp\u003eIn order to evaluate the effect of hucMSC-exos on the histopathological changes in PU mice, hematoxylin and eosin (HE) staining and Masson staining were conducted. The results demonstrated that hucMSC-exos decreased the wound scar areas and regular epidermal and dermal structures in PU mice compared with the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA); and densely packed with collagen fibers in the dermal layer, and arranged in parallel in the epidermis were observed following hucMSC-exos treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). RT-qPCR results demonstrated that hucMSC-exos decreased the expressions of α-SMA, CD34, collagen I, and collagen III in PU mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD); and further immunochemistry results demonstrated that the expression of α-SMA and CD34 were also decreased in the PU mice following the administration of hucMSC-exos (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These data demonstrated that the beneficial effects of hucMSC-exos treatment on wound healing and skin tissue regeneration in PU mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4 The underlying mechanisms of hucMSC-exos treatment on PU mice\u003c/h2\u003e\u003cp\u003eThe results of immunohistochemical analysis demonstrated that expression of HMGB1 was high in epidermal tissues of the PU mice, and the expression of HMGB1 was significantly decreased following the administration of hucMSC-exos (Fig.\u0026nbsp;6C). In addition, the results of the western blotting analysis demonstrated a decline in the expressions of HMGB1 and α-SMA following the treatment with hucMSC-exos (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results demonstrated that the HMGB1/α-SMA axis regulated the therapeutic effect of hucMSC-exos in PU mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePUs are a type of skin lesion that has been identified as the primary cause of I-R, which is associated with an anti-inflammatory phase characterized by severe defects in cell-mediated immunity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It is evident that a multitude of many cellular elements and intricate molecular interactions are involved in this inflammatory reaction. hucMSC-exos, which contain various bioactive molecules, including mRNAs and microRNAs, have been shown to facilitate intracellular communication [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Moreover, these exosomes have been demonstrated to exert significant paracrine effects on wound repair, promoting cell migration and metastasis while concomitantly attenuating the inflammation associated with wound healing [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this article, we isolated the exosomes from hucMSCs firstly, further confirmed that hucMSCs-exos could be uptake by PU mice, and the data demonstrated that the administration of hucMSCs-exos has the capacity to impede cutaneous wound healing and to ameliorate the histopathological changes in PU mice. These results indicated that hucMSC-exos had the good drug safety and therapeutic effects on PU.\u003c/p\u003e\u003cp\u003eIn normal skin, CD34 is primarily located in endothelial cells, dermal dendritic cells, and perifollicular cells. It has been demonstrated that CD34\u003csup\u003e+\u003c/sup\u003e cells have a significant role in the process of wound healing, they have the capacity to enhance the epithelial regeneration, mechanical support, intercellular communication, and immune regulation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. is a protein that plays a pivotal role in maintaining the structural integrity and elasticity of various bodily tissues, including the skin, bones, and tendons. It is also involved in crucial processes such as organ and tissue development, cell migration, proliferation, and differentiation, wound healing, and tissue remodeling. Among the various types of collagen, collagen I and collagen III are particularly prominent, accounting for the highest proportion [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. α-SMA is commonly found in fibroblasts, smooth muscle cells, and muscle cells. It is significantly upregulated shortly after the inflammatory phase of skin trauma and plays a crucial role in the formation of the extracellular matrix [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It has been demonstrated that the function of MSC-derived exosome varies at different stages of the wound-healing process. In the initial phase of wound healing, the levels of type III collagen, type I collagen, and CD34 increased following the administration of MSC-derived exosome therapy. During the remodelling phase, the expression levels of type III collagen, type I collagen, and α-SMA decreased [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this article, the data demonstrated that the expressions of type III collagen, type I collagen, and α-SMA were decreased after the treatment of hucMSC-exos for for a period of 14 days. These data demonstrated that hucMSC-exos exhibited effective tissue repair function and reduced scar formation in PU mice.\u003c/p\u003e\u003cp\u003eHMGB1 is a chromatin-bound nuclear protein that can also act as a cytokine under stress conditions or during cell death. has been identified as a pivotal factor in the induction of sterile immunity, with its release by ischemic cells being a crucial element in the activation of the inflammatory pathway [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The release of HMGB1 from the cell is a significant event in the injury process, as it plays a crucial role in the inflammatory response, akin to that of a cytokine. Furthermore, HMGB1 has been demonstrated to induce fibrosis and scar formation, while the inhibition of HMGB1 has been shown to impede fibroblast migration and collagen synthesis, thereby significantly reducing scarring [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our data demonstrated that the high expression of HMGB1 was observed in the dermis layer of PU mice, and that the expression of HMGB1 was significantly decreased by hucMSC-exos. This was further confirmed by RT-qPCR and western blotting. These data demonstrated HMGB1 may may have a role in regulating the treatment of PU with hucMSC-exos.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, we confirmed that hucMSC-exos had the therapeutic effect on the promotion of tissue re-epithelialization and vascularization of PU by regulating HMGB1 and α-SMA, which may provide a promising strategy for the treatment of PU.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFY, MHG, JZY and LY contributed to data analysis and interpretation, data collection, literature review, and drafted the manuscript. FRL and LY contributed to the conception and design of the study. FY, and MHG contributed to the MSC production and characterization. FY, MHG, JZY, FRL and LY participated in the data collection and interpretation. All authors critically reviewed and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have made substantial contributions to the conception or design of the work and to revising and drafting the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Translational Medicine Collaborative Innovation Center and Plastic and Aesthetic Department in Shenzhen People\u0026rsquo;s Hospital. This study was supported financially by the Hospital young and middle-aged research backbone training project (1033).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was approved by the Ethics Committee of the Medical Ethics Committee of Shenzhen People\u0026rsquo;s Hospital. The medical ethical number: LL-LK-201928\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll materials are available from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSun J, Wang ZJ, Wang XR (2018) Suppression of LRRC19 promotes cutaneous wound healing in pressure ulcers in mice. 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This condition can result in damage, ulceration, and even necrosis. It serious threat to the patient's life and health, with the potential to result in fatal consequences. (MSCs) have been shown to secrete exosomes, termed MSC-exos, which range \u0026nbsp;in size from 40 to 160 nm, and have been found to contain DNA, lipids, and proteins, and have been implicated in the treatment of various diseases. However, the effect of hucMSC-exos on the PU and the underlying mechanisms remain not to be fully elucidated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: The present study investigates the effects of exosome isolation from hucMSCs on the mouse PU model. Following the administration of hucMSC-exos, the healing of the wound and the histopathological changes in the mice were evaluated by HE staining and Masson staining; the collagen mRNA levels in the tissues were analyzed by quantitative real-time polymerase chain reaction (qRT–PCR); the expressions of HMGB1, α-SMA, and CD34 were examined by western blotting and immunohistochemistry to investigate the potential mechanisms by which the therapeutic effect of hucMSC-exos on PU is achieved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The results of HE staining and Masson staining demonstrated that hucMSC-exos could alleviate the histopathological symptoms of PU. qRT–PCR results demonstrated that hucMSC-exos decreased the levels of collagen I, collagen III, TGF-β, and HGMB1 in PU mice, the results of western blotting demonstrated that the expressions of HMGB1, SMA, and CD34 could also be decreased by the administration of hucMSC-exos.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe present study demonstrated that hucMSC-exos alleviated the symptoms of PU by regulating HGMB1 and α-SMA. which demonstrated that the therapeutic potential of hucMSC-exos in PU.\u003c/p\u003e","manuscriptTitle":"Effect of hucMSC-derived exosomes on the pressure ulcers in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 10:25:50","doi":"10.21203/rs.3.rs-7005414/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":"b061e770-c4eb-41f9-a013-1ece258da973","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-05T22:08:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-31 10:25:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7005414","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7005414","identity":"rs-7005414","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}