Hyaluronic acid stimulation of induced MSCs produces extracellular vesicles with enhanced healing for skin burn wounds

Hyaluronic acid stimulation of induced MSCs produces extracellular vesicles with enhanced healing for skin 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 Hyaluronic acid stimulation of induced MSCs produces extracellular vesicles with enhanced healing for skin burn wounds Minyoung Jung, Hyun Geun Oh, Seul Ki Lee, Eun A Kim, Haedeun You, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4821606/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 Skin injuries occur for various reasons during whole life. Some chronic wounds could cause an impaired wound healing process characterized by wound hypoxia, high levels of oxygen radicals, elevated levels of matrix metalloproteases, delayed cellular infiltration and granulation tissue formation, reduced angiogenesis, decreased collagen synthesis and organization. In this study, we report the EVs from hyaluronic acid-primed iMSCs (HA-iMSC-EVs) accelerating wound healing and regenerating damaged tissues by inducing the various growth factors in the thermal injury of mice. Methods EVs were collected from iMSCs primed with HA (HA-iMSC-EVs) or without HA (iMSC-EVs) and were isolated using TFF systems. Both EVs analyzed the characteristics. We investigated the proteome of HA-iMSC-EVs using the protein set ontology analysis and protein-protein interaction network. To evaluate the effect of HA-iMSC-EVs on the oxidative stress-induced wound healing delayed model, we assessed the effect of EVs on cell viability, cell migration rate, and the mRNA expression of growth factors using a hydrogen peroxide-exposed HDF model. In addition, we observed elastin and collagen expressions using an ICC staining in the HDF model. In thermal burn wound mice (BALB/c), we compared the effect of EVs in wound closure rate and histological analysis, including expression of elastin, collagen, α-SMA, and CD31. Results HA-iMSC-EVs exhibited typical EV characteristics, including size distribution, markers, and surface protein expression. In GO term analysis, HA-iMSC-EVs increased the proteins associated with ECM, including collagen biosynthesis and elastin fiber formation. In hydrogen peroxide exposed HDF models, HA-iMSC-EVs notably increased cell viability and migration activity. Furthermore, HA-iMSC-EVs increased RNA expression of VEGF, IGF1 , and HGF and decreased IL-6 mRNA expression compared to the PBS group. Elastin and collagen expression in the HA-iMSC-EVs group were also significantly increased. In burn-injured mice, HA-iMSC-EVs accelerated wound closure and enhanced histological recovery. HA-iMSC-EVs increased collagen and elastin density on the upper dermis and decreased α-SMA expression. Additionally, HA-iMSC-EVs promoted the capillary density in the dermis. Conclusions Our results suggest that HA-iMSC-EVs accelerated the recovery from burn wound by providing ECM composition signal and regulating growth factors. Our strategy may contribute to the development of alternative treatment option for burn wounds. Trial registration : Not applicable. Extracellular vesicles burn wound chronic wound extracellular matrix growth factors. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Burn injuries remains a serious public health issue with a high prevalence and fatality rate. According to WHO estimates, 180,000 of the 11 million burn injuries occur annually worldwide are fatal [ 1 ]. The breakage of skin integrity provokes infection and compromised skin function, thus immediate treatment is needed. In severe burn injury, skin regrowth starts from marginal area, which makes the chance of infection high. The pathological feature from this injury include metabolic changes [ 2 ], immune responses [ 3 ], which can lead to shock and multiple organ failure [ 2 ]. Moreover, the overall quality of life and psychological health can be profoundly impacted by burn injuries [ 4 ]. The current gold standard for managing burn wounds that are extensive or severe is autologous skin grafting followed by early excision [ 5 , 6 ]. Despite advances in technologies, managing and treating burn wounds still remains significant obstacles. polymorphonuclear neutrophils (PMNs) immediately invade the lesion and release large amounts of reactive oxygen species (ROS) that cause endothelial cell and skin damage. ROS increases capillary permeability and promote edema formation in the zone of stasis [ 7 , 8 ]. The overproduction of free radicals also impairs the production and release of growth factors such as EGF, TGF-β, and VEGF, causing delayed wound healing [ 9 ]. Thus, growth factors have been given attention as the key molecules regulating wound repair and regeneration by their therapeutic role in the pathophysiology of chronic wounds [ 10 , 11 ]. However, their translation to the clinic has been limited because they have a short in vivo half-life due to their low stability and restricted absorption rate [ 12 ]. Regenerative wound healing uses emerging biomedical research technologies, including targeted drug/growth factor delivery, smart wound dressings, bioactive biomaterial matrices, gene therapy, and stem cell therapy [ 13 , 14 ]. The ideal healing of skin wounds involves regenerating damaged skin tissue while preventing the progression of chronic wounds and reducing scar formation (hypertrophic scar or keloid). Although the enormous impact of chronic wounds and fibrosis on human health and increasing prevalence and cost, chronic wounds and scar formation remain an unmet treatment area [ 15 ]. The feasibility of stem cell-based treatments for burn injury have been shown in recent preclinical studies, and this field is one of the most active areas that are under clinical investigation [ 16 , 17 ]. In particular, many preclinical studies reported that various types of MSCs reduced fibrosis and repaired wounds [ 18 ]. It was also shown that MSCs promote wound healing via their paracrine function rather than their ability to differentiate into skin cells [ 19 , 20 ]. However, the therapeutic outcome of MSCs is not optimal, mostly due to multiple obstacles such as low survival in vivo, thrombosis, and tumorigenesis. Additionally, the therapeutic potential of MSCs reduces and they undergo cellular senescence during long term culture [ 21 , 22 ]. Extracellular vesicles (EVs) are lipid bilayered-particles that are essential for cell-to-cell communication because they carry certain biomolecular cargos that are necessary for tissue homeostasis and recovery. Importantly, EVs derived from MSCs (MSC-EVs) have several advantages over MSCs, including abundant resources, low immunological rejection, and the ability to be reengineered or combined with other biomaterials [ 23 ]. Moreover, different strategies (e.g., genetic modification or cell priming) can be implemented to modify the biomolecular cargo of EVs depending on the purpose [ 24 ]. Compared to other approaches, small molecule preconditioning/priming strategies have several advantages, such as being quick and simple to use and having well-established underlying processes [ 25 , 26 ]. Hyaluronic acid (HA) is an essential component of the ECM that has numerous functions including extracellular matrix function, cell migration, and wound repair [ 27 – 29 ]. In this study, we assessed whether EVs from HA-primed iMSCs (HA-iMSC-EVs) can accelerate the recovery of burn injury in mice skin. Relevant in vitro experiments were also conducted to determine the mechanism of their function. Materials and methods EV isolation For EV isolation, iMSCs were generated from induced pluripotent stem cells (STEMCELL Technologies Inc., Vancouver, Canada) as described in our previous study [ 30 ], and iMSCs were established as a working cell bank at the R&D center of Brexogen Inc. (Seoul, Rep. of Korea). Briefly, established iMSCs were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; HyClone, Chicago, IL, USA) supplemented with 15% fetal bovine serum (FBS; HyClone) and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in a humidified incubator containing 5% CO 2 . At 90% confluence, cells were detached using TrypLE Express (Thermo Fisher Scientific) and seeded at a density of 10,000 cells/cm 2 . The next day, the cells were stimulated with or without 40 µg/mL HA (Sigma-Aldrich, St Louis, MO, USA) for 24 h. After being washed with Dulbecco’s Phosphate Buffered Saline (HyClone), cells were cultured in phenol red-free DMEM (Gibco, Waltham, MA, USA) supplemented with 15% EV-depleted FBS. After 3 d of incubation, the culture medium was harvested and centrifuged at 300 × g for 5 min and 2,000 × g for 30 min to remove cells and debris. The supernatant containing HA-iMSC-EVs was filtered using a vacuum filter (Merck, Darmstadt, Germany) and then was diafiltered with Dulbecco's Phosphate-Buffered Saline (DPBS, Gibco) by tangential flow filtration (TFF) systems (Sartorius AG, Göttingen, Germany) (Additional file 1: Figure S1 , A). Nanoparticle tracking analysis The particle size and concentration of EVs were measured Zetaview®BASIC NTA-Nanoparticle Tracking (Particle Metrix, Inning am Ammersee, Germany). The values for the standard controls were set as follows; Sensitivity: 80, Frame Rate: 30, Shutter: 100, Temperature: 23°C. Immunoblotting To characterize surface proteins of EVs, western blotting was performed as previously described [ 31 ]. Briefly, EVs were lysed in RIPA lysis buffer with protease inhibitors (Thermo Fisher Scientific). Protein concentration was measured using the Bradford Assay Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Proteins were loaded and separated on precast polyacrylamide Mini-PROTEAN TGX gels (Bio-Rad Laboratories, Hercules, USA) and transferred to PVDF membranes (Bio-Rad Laboratories). The membranes were incubated overnight with primary antibodies at 4°C. Antibodies against CD63 (Abcam, Cambridge, UK) and TSG101 (Invitrogen, Waltham, MA, USA) were used as the primary antibodies. Flow cytometry HA-iMSC-EVs analysis was performed using human MACSPlex Exosome Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Flow cytometric analysis was conducted using an Attune NxT flow cytometer (Thermo Fisher Scientific), as previously described [ 31 ]. Proteome profiling and bioinformatic proteome analyses To compare protein sets of iMSC-EVs and HA-iMSC-EVs, liquid chromatography with tandem mass spectrometry (LC/MS–MS) analysis was employed in CellKey Co., Ltd (Seoul, Rep. of Korea). Briefly, before LC/MS-MS analysis, proteins are extracted from each three EV batches using RIPA lusis buffer (Thermo Fisher Scientific). After protein digestion by trypsin, prepare the desalting peptide fraction for LC/MS-MS. An EASY-Spray PepMap Neo C18 column (Thermo Fisher Scientific) was used with the mobile phase consisting of buffer A (0.1% formic acid in water) and buffer B (0.1% formic acid in acetonitrile). A portion was injected at 300 nL/min into the LC system. MS–MS analysis was carried out on a Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer in the nano ESI positive ion mode. Tryptic peptides were identified and quantified using Proteome Discoverer (Thermo Fisher Scientific). A database including 20,307 proteins from UniProt was used to identify proteins in the samples. The identification of proteins was performed with 1.0% of false discovery rate (FDR). Normalized peptide abundances were used for the label-free quantification of proteins. The fold change (Log 2 FC) of protein abundance values cutoff was set at 1.0 (two-fold up-regulation, p-adj < 0.05, Additional file 2: Table S2 ). Protein set ontology analysis was performed using the DAVID at an FDR q-value cutoff of 0.05 ( https://david.ncifcrf.gov/ ). The protein sets subjected to the Markov Cluster Algorithm (MCL) cluster analysis and protein-protein interaction (PPI) network with confidence of 0.7 using STRING 12.0 ( https://string-db.org/ ). Hydrogen peroxide treatment and cell viability measurement using MTS assay Human dermal fibroblasts (HDFs; ScienCell Research Laboratories, Carlsbad, CA, USA) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; GIBCO® Invitrogen, Carlsbad, CA, USA) containing penicillin (100 IU/ml), streptomycin (100 µg/ml), and 10% fetal bovine serum (FBS; GIBCO®) at 37℃ in a humidified 5% CO 2 incubator. HDFs at passages 8–10 were manipulated for in-vitro assay. Cells were cultured in 96-well plates (5,000 cells/well) and grown overnight at 37˚C. Cells were incubated with various concentrations (0.16-5 mmol/L) of hydrogen peroxide for 3 hours at 37˚C under 5% CO 2 . Following exposure to mechanical strain and hydrogen peroxide, media was exchanged with the serum-free condition and treated with HA-iMSC-EVs at 0, 5, 10, 20, 40, 80 µg/ml. After 24 hours of incubation, MTS solution (CellTiter kit; Promega, Madison, WI, USA) was added to each well and incubated for one hour. The optical density at a wavelength of 490 nm was detected. Cell migration assay Cell migration assay was conducted using a wound healing assay Kit (Abcam) according to the manufactured protocol. HDFs were seeded in 12-well plates (5×10 4 cells/well) assembled with inserts to make the gap (empty area) and 24 hours of cultivation, the inserts were removed, then cells were rinsed with DPBS. Following exposure to hydrogen peroxide for 3 hours, each plate added HA-iMSC-EVs or control vehicles with the DMEM media including 5% exosome depleted FBS, and cultivated them under standard cell culture conditions. At 0 h and 30 h, the wound healing was recorded by a microscope (Leica, Germany). Image J software was used to quantitatively evaluate the wound healing rate. Quantitative RT-PCR Cells were harvested for total RNA extraction using a TRIzol® (Invitrogen) according to the manual protocol. Complementary DNA (cDNA) was then synthesized from 500 ng total RNA per sample using an AccuPower®RT PreMix kit (Bioneer Inc. Seoul, Rep. of Korea), followed by qPCR using a Power SYBR Green PCR Master Mix kit (Applied Biosystems). The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as an internal control and the qPCR primers used are listed in additional file 1: Table S1 . Immunocytochemistry The cultured cells in 12-well plates were washed with DPBS and fixed in cold 100% methanol at -20℃. The cultures were incubated with 10 mg/ml of a polyclonal antibody to tropoelastin (Elastin Products, Owensville, MI) or polyclonal antibody to collagen type I (Abcam). Cultures were then incubated with the respective fluorescein-conjugated secondary antibodies. Nuclei were counter stained with DAPI (Invitrogen, Waltham, MA, USA). The stained cells were observed under a Nikon Eclipse Ti2-U fluorescent microscope (Nikon, Tokyo, Japan). Animal experiments All animal experiments were conducted in compliance with the approved animal protocols by IACUC of Asan Medical Center, Seoul, Korea (Approval No. 2018-12-284). 12-week-old female BALB/c mice (20-25g) were assigned randomly into three groups as follows: PBS group (n = 5), iMSC-EVs group (n = 5), and HA-iMSC-EVs group (n = 5). The animals were kept under 25 ± 3 ℃ temperature and relative humidity of 30–70% in 12 h light/dark cycle with standard food and water were available ad libitum . The thermal injury model was developed by modifying the method of Tavares Pereira et al. [ 32 ]. Briefly, fifteen mice were anesthetized with Zoletil (30 mg/kg, Virbac, Carros, France) and Rompun (10 mg/kg, Bayer, Leverkussen, Germany). Following anesthesia, the dorsal areas of the mice were shaved. An aluminum plate (diameter = 10 mm) preheated at 100°C for 15 seconds was placed directly onto the depilated area to create symmetrical burns. Following the burn injury, burn wounds were covered with hydrocolloid dressing (DuoDerm®, ConvaTec, Berkshire, UK), then 100 µL HA-iMSC-EVs (1 mg/mL), iMSC-EVs (1 mg/mL), or PBS was used subcutaneously administrated. To minimize the variance that can arise from the order of treatments, administration was performed in following order: 1) iMSC-EVs, 2) HA-iMSC-EVs, and 3) PBS. The closure of burn wound was examined for 15 days (Additional file 1: Figure S1 , B). The sample size was estimated using the online Sample size calculator program v1.061 for ANOVA analysis ( https://homepage.univie.ac.at/robin.ristl/samplesize.php ) based on the wound closure rates from the pilot study (alpha = 0.05, power = 0.8). The study was reported in line with the ARRIVE guidelines 2.0, with additional supporting documents provided in the supplementary materials. Histological analysis The mice were euthanized by CO 2 inhalation and the skin tissues were harvested at day 15 after thermal injury for burn wounds. Skin specimens were fixed in 4% paraformaldehyde, and paraffin embedded. H&E staining and Verhoeff van Gieson staining were performed according to routine protocols. And whole slides were scanned using Panoramic digital slide scanner (3DHISTECH), generating a TIFF file. The scanned images were evaluated blindly by two investigators. The evaluation range was limited to the dermis at equal distances from the basal layer of the epidermis in the wounded lesion. The region of interest (ROI) was set to the same size for each slide image at 200x magnification, five ROI values were measured for each slide, and the average value was calculated. The density of elastin and collagen fibers of each ROI was measured with Image J software program. Immunohistochemical staining was performed using a primary antibody against mouse CD31 (Abcam, Cambridge, UK), or mouse alpha smooth muscle actin (α-SMA, Abcam). And histological images were conversed by whole slide scanner. The α-SMA density was quantified using Image J software program. Statistical analyses Statistical analyses were performed using SPSS (version 18.0 for IBM, Chicago, IL, USA). For comparisons involving three or more groups, one-way analysis of variance (ANOVA) was performed followed by Tukey’s post hoc test. Data are expressed as means ± standard error (SEM), and values with p < 0.05 were considered statistically significant. Results Characterization of HA-iMSC-EVs To confirm the characteristics of the iMSC-EVs and HA-iMSC-EVs, we measured the size of the EVs using NTA. The average diameters of iMSC-EVs and HA-iMSC-EVs were 130.4 and 136.9 nm, respectively (Fig. 1 A). Both the EV types expressed CD63 and TSG101 (Fig. 1 B). Flow cytometry revealed that iMSC-EVs and HA-iMSC-EVs tested positive for CD9, CD63, and CD81, which are typical extracellular vesicle surface markers, but negative for stage-specific embryonic antigen-4 (SSEA4) and CD45 (Fig. 1 C). These results indicate that HA-iMSC-EVs met the general characteristics of EVs derived from cultured cells [ 30 , 33 ]. The HA-iMSC-EVs proteome is associated with ECM construction-related functions To determine the effect of HA preconditioning on the cargo protein profile, proteomic analysis was conducted by LC/LC-MS. A total of 821 proteins were identified in iMSC-EVs and HA-iMSC-EVs (Additional file 1: Figure S2 ). Among the 579 proteins identified in HA-iMSC-EVs, 422 proteins (72.9%) overlapped with those in iMSC-EVs. GO and KEGG analyses were performed on the protein sets that showed more than a 2-fold increase in HA-iMSC-EVs compared to those from iMSC-EVs from these overlapping genes (Fig. 2 A). A total one hundred and fifty-nine proteins increased in HA-iMSC-EVs by HA preconditioning were analyzed using the DAVID program to identify the GO terms. The proteins set increased in HA-iMSC-EVs were related to the function of ECM, such as 'regulation of collagen fibril organization' (GO:190426, FDR = 1.88E-02), 'extracellular matrix organization' (GO:0030198, FDR = 1.91E-15) and 'collagen biosynthetic process' (GO:0032964, FDR = 6.85E-03). Additionally, the protein set of HA-iMSC-EVs was related to ECM-receptor interaction (hsa04512, FDR = 3.39E-07), tight junction (hsa045320, FDR = 2.53E-03) and PI3K-AKT signaling (hsa04151, FDR = 1.16E-04) in KEGG pathway analysis. Through the STRING program, the proteins set was subjected to cluster analysis with Markov Cluster Algorithm. Then, the PPI network of the largest cluster containing the set of ECM-related proteins was analyzed (Fig. 2 B & Additional file 1: Figure S3 ). As results, HA-iMSC-EVs were enriched with proteins related to collagen synthesis (FDR = 7.66E-34) and elastic fiber formation (FDR = 9.22E-12). KEGG pathway analysis showed that the proteins of HA-iMSC-EVs are involved in TGF-β (FDR = 3.00E-03) and PI3K-AKT (FDR = 2.59E-10) signaling pathways. HA-iMSC-EVs enhance cell viability and migration of HDFs undergoing oxidative stress We first examined the effect of H 2 O 2 on the viability of HDFs by MTS assay. As presented in Fig. 3 A, the viability of HDFs was reduced under 0.63, 1.25, 2.5, and 50 mM of H 2 O 2 (p < 0.001). We used 0.63 mM of H 2 O 2 for subsequent studies. HDFs were exposed to H 2 O 2 for 3 hours, and then incubated with serum-free medium containing iMSC-EVs or HA-iMSC-EVs for 24 hours. As shown in Fig. 3 B, the viability was significantly higher in HDFs treated with 20 or 40 µg/mL HA-iMSC-EVs compared with those treated with PBS (p < 0.0001). In contrast, no change was observed in iMSC-EVs- or HA-treated cells. Consistently, HA-iMSC-EVs stimulated the migration of HDFs under oxidative stress compared with PBS-treated cells (p < 0.05). No effect was observed in iMSC-EVs- or HA-treated cells (Figs. 3 C and 3 D). HA-iMSC-EVs decrease inflammatory cytokines expression and enhance growth factor expression in HDFs undergoing oxidative stress RT-qPCR analysis showed that hydrogen peroxide increased the mRNA expression of pro-inflammatory cytokines (i.e., TNF-α, IL-1β , and IL-6 ) in HDFs. HA-iMSC-EVs caused a slight reduction of the expression of TNF-α and IL-1β as compared with PBS. A significant decrease of IL-6 was found in HA-iMSC-EVs compared with PBS-treated cells (Fig. 4 A). No change in the mRNA expression of growth factors was observed after hydrogen peroxide treatment in HDFs. HA-iMSC-EVs augmented the mRNA expression of growth factors ( TGF-β1 , VEGF , IGF1 , EGF , and HGF ) (Fig. 4 B, p < 0.05). No change was observed in iMSC-EVs- or HA-treated HDFs. HA-iMSC-EVs increase type 1 collagen and elastin expression in HDFs undergoing oxidative stress As shown in Fig. 5 , hydrogen peroxide decreased the protein expression of type I collagen and elastin. Both iMSC-EVs and HA-iMSC-EVs was able to augment the expression of Type 1 collagen (p < 0.01), and such increase was more significant in HA-iMSC-EVs compared with iMSC-EVs (Fig. 5 C) (p = 0.048, t-test). Additionally, HA treatment led to an increase in type I collagen expression, which was comparable to the levels observed with iMSC-EVs (p = 0.088, t-test). Similarly, both iMSC-EVs and HA-iMSC-EVs enhanced the protein expression of elastin in hydrogen peroxide-exposed HDFs. However, such increase was more significant in HA-iMSC-EVs compared with those from iMSC-EVs (p = 0.015, t-test), which was comparable to those from intact cells (Fig. 5 D). No increase in the expression elastin was noted in HA-treated cells. Interestingly, many elastin fibrils were observed in the HA-iMSC-EVs group, similar to those observed in intact cells (Fig. 5 B). In contrast, only accumulated tropoelastin was observed in the iMSC-EVs group. HA-iMSC-EVs improves wound healing in burn injury of mice We next evaluated whether HA-iMSC-EVs had a greater effect in the rate of wound closure after burn injury. In iMSC-EV, there was a minor increase in wound closure; however, no significant increase against PBS was found. In HA-iMSC-EVs-treated animals, however, the wound closure was significantly enhanced compared with those from animals receiving PBS. Even on Day 9, 80.9% of the burn wound was closed in the HA-iMSC-EVs group, which was higher than those found in the iMSC-EVs group on Day 13. On Day 15, no change was observed in the wound closure rate among three groups (Fig. 6 A, B). HA-iMSC-EVs enhance dermal matrix formation and reduce α-SMA Microscopic analysis showed that both types of EVs enhanced the formation dermal matrix; however, such change was more prominent in HA-iMSC-EVs compared with iMSC-EVs (Fig. 7 A). Quantitative measurement revealed that the increase in the positive area that reacted with collagen antibody was observed only in HA-iMSC-EVs-treated animals (p < 0.01). Both iMSC-EVs and HA-iMSC-EVs promoted the elastin density, however, such increase was more significant in HA-iMSC-EVs (p < 0.05 and p < 0.01 in iMSC-EVs and HA-iMSC-EVs, as compared with PBS) (Fig. 7 B, C). Neovascularization is essential for wound healing, as maladaptive vascularization is a common feature of chronic wounds [ 34 ]. Capillaries created during wound healing regress as granulation tissue converts into mature scar tissue. Eventually, the number of vessels returns to a level close to that observed in uninjured skin [ 35 ]. As shown in Fig. 7 D and 7 E, a significant reduction in CD31-positive vessels was found only in HA-iMSC-EVs-treated mice (p < 0.05 vs. PBS); no effect was observed in iMSC-EVs. The expression of α-SMA is responsible for contraction of wound during early stage and eventually disappears [ 36 ]. However, its persistent expression can be found in fibrotic scars [ 37 , 38 ]. The expression of α-SMA was decreased only in HA-iMSC-EVs as compared with PBS (p < 0.01). In contrast, its diffuse expression was observed in iMSC-EVs, which was similar to those in PBS-treated group (Fig. 7 A, B). Discussion Exosomes control various biological processes, and they have showed potential in treating diseases including cardiovascular, immune, and neuronal diseases [ 39 ]. The horizontal transfer of biomolecules by exosomes was first demonstrated in 2007, whereby exosomal mRNA can be translated upon entering receiving cells [ 40 ]. Following this report, studies have demonstrated that exosomes carry lipids, proteins, microRNAs, lncRNAs, and circRNAs, which are involved in regulation of inflammatory response, cell proliferation, migration, angiogenesis, and ECM remodeling [ 41 ]. Importantly, these events are essential for successful wound healing, and maladaptive repair events can lead to chronic wound development. The aim of the present study was to examine whether the HA stimulation of iMSCs can produce EVs that have enhanced function in the recovery of skin burn injury. HA-iMSC-EVs had proteins associated with the maintenance of skin integrity (ECM production and organization, TGF-β signaling, and PI3-AKT signaling etc.). Under H 2 O 2 -induced oxidative stress, the migration and viability of HDFs were increased by HA-iMSC-EVs compared with those treated with iMSC-EVs. The elevated level of IL-6 expression by H 2 O 2 was reduced only by HA-iMSC-EVs. Additionally, the mRNA expression of key growth factors was increased only by HA-iMSC-EVs. Also, the decrease of elastin and collagen protein expression by oxidative stress was reversed by both iMSC-EVs and HA-iMSC-EVs, but its effect was more significant in HA-iMSC-EVs. In burn-injured mice, accelerated wound closure was observed only in animals that received HA-iMSC-EVs. The expression of collagen in skin tissue was enhanced only by HA-iMSC-EVs. Both iMSC-EVs and HA-iMSC-EVs stimulated elastin production, with the latter more significant. The recovery of capillary density as well as reduction of α-SMA expression in dermal layer was observed only in animals that received HA-iMSC-EVs. Together, these results suggest that HA treatment of iMSCs produce EVs having enhanced potential the recovery of skin tissue after burn injury by stimulating skin cell proliferation, growth factor production, vessel formation, and ECM production in the lesion. Skin is the largest organ of the body and plays multiple roles including sensation, body heat regulation, protection, and host defense [ 42 ]. Skin is prone to various injuries including trauma, surgery, chronic disease, and burns. In healthy individuals, injured skin recovers through the four stages of wound healing: hemostasis, inflammation, proliferation, and remodeling [ 43 ]. On the other hand, some chronic wounds in diabetes or ischemia can cause an impaired wound healing process characterized by hypoxia, oxygen radicals, matrix metalloproteases, granulation tissue formation, reduced angiogenesis and decreased collagen synthesis and organization [ 44 ]. Following an acute burn injury, a strong inflammatory response occurs, which is characterized by neutrophil and macrophage recruitment to the injury site [ 12 ]. The innate immune cells and injured tissue release a wide range of inflammatory cytokines and growth factors (e.g., IL-1, IL-6, TGF-β, EGF, VEGF). When the production and secretion of these cytokines become excessive, wound closure can be delayed or the injury can progress to chronic stage [ 45 ]. Previous studies showed that MSC-EVs can block inflammation of skin burn injury; Li et al. showed that exosomal miR-181c from human umbilical cord-derived MSCs inhibited inflammation by suppressing Toll-like receptor 4 (TLR4) signaling in skin burn injury model as well as those in LPS-stimulated macrophages [ 46 ]. More recently, Liu et al. conducted single cell sequencing analysis from the peri-wound skin of mice that had undergone full-thickness excision injury, and found that exosomes from human umbilical cord MSCs led to an increase of the proportion of M2 macrophages and neutrophils [ 47 ]. Since inflammatory events can affect the degree of wound closure and scar formation [ 48 ], it would be needed to elucidate whether HA-iMSC-EVs can also repress the inflammation by regulating macrophage polarization at the early phase after injury. Growth factors play crucial role in the wound healing process by regulating immune cells, promoting migration and proliferation of dermal cells (e.g., epithelial cells and fibroblasts), collagen synthesis, and differentiation [ 49 ]. For example, it was demonstrated that EGF is crucial for improvement of re-epithelialization [ 50 ], and that IGF-1 accelerates wound healing by promoting angiogenesis [ 51 ]. Also, HGF was shown to augment neovascularization, re-epithelialization of skin wounds, and granulation tissue formation [ 52 ]. However, due to the complexity of molecular pathways and wound chronicity, the local application of a single exogenous growth factor is insufficient to improve burn wounds [ 53 ]. Therefore, delivering a combination of growth factors using methods with high diffusion efficiency and bioavailability into the burn lesions is important for wound healing. Previous study showed that HA-iMSC-EVs enhance human umbilical vein endothelial cells (HUVEC) tube formation and angiogenesis [ 19 ]. Our study demonstrated that HA-iMSC-EVs increased the expression of various growth factors in skin fibroblasts. Thus, it is likely that HA-iMSC-EVs accelerated wound healing by improving re-epithelialization and neovascularization. ECM deposition is the last phase during wound healing process, and its failure can lead to chronic wound or excessive scar formation. Collagen and elastin are the two most essential ECM proteins in skin [ 54 ], and collagen accounts for 50–90% of the dermal matrix. In line with our findings, Kim et al. reported that human umbilical cord-derived MSCs enhanced the cell migration as well as the synthesis of collagen and elastin in HDFs [ 55 ]. Furthermore, it has been shown that exosomes produced from human-induced pluripotent stem cell-derived MSCs (hiPSC-MSCs) promoted the expression of elastin, collagen I, and collagen III in HDFs [ 56 ]. In contrast, other studies demonstrated that MSC-derived exosomes inhibit the excessive production of ECM as well as fibroblast-to-myofibroblast transition, preventing scar formation. For example, it was shown that ADSC-derived exosomes reduced excessive scar formation by keeping fibroblasts from developing into myofibroblasts as well as controlling the ratios of TGF-β3/TGF-β1, collagen III/collagen I, and MMP3/TIMP1 [ 57 ]. We demonstrated that excessive expression of α-SMA was inhibited by HA-iMSC-EVs, which might have contributed to inhibiting excessive scar formation by the reducing the number of myofibroblasts, while increasing structural integrity (e.g., by collagen production) in dermis. Elastin is responsible for tensile strength, providing structural integrity of skin. Mature, functional elastin is assembled from tropoelastin monomers through coacervation, cross-linking, and deposition [ 58 ]. However, the synthesis of new tropoelastin stops after the neonatal period [ 59 ], and insufficient elastic fiber network contributes to the reduced elasticity and resilience of the mature scar [ 60 ]. The restoration of an immediate and functional elastic fiber is, therefore, critical to regain complete skin function after injury, and it is needed to develop novel strategy to increase elastin to restore skin function. Our results demonstrated that the increase of elastin expression was higher in HA-iMSC-EVs compared with iMSC-EVs in the recovered skin. In the fibroblasts, elastin fibrils were observed only in the HA-iMSC-EVs. In support of this findings, bioinformatic analysis showed that HA-iMSC-EVs have proteomic profile that are related to ECM composition, and that enriched proteins in HA-iMSC-EVs were related to elastic fiber formation and collagen biosynthesis. One of the key protein families required for tissue remodeling after injury is matrix metalloproteinases (MMPs) [ 61 ]. It was shown that hADSC-Exos activated MAPK pathway, stimulating the production of MMP-3 and TIMP-1, which led to enhanced ECM remodeling [ 57 ]. Another study demonstrated that miRNA-21 in hADSC-Exos promoted MMP-9 levels, while decreased TIMP2 as well as TGF-β1, thus reducing the formation of wound scars [ 62 ]. Zhang et al. reported that hADSC-exos augmented MMP-1 expression and downregulated α-SMA expression, and that promoted collagen deposition, improving dermal thickening in full-thickness incision wound model [ 63 ]. Thus, other in-depth studies such as the role of HA-iMSC-EVs in regulating MMPs/TIMPs and myofibroblast activation are needed to further examine detailed mechanisms how ECM deposition was increased in dermal layer. We observed that the increase of IL-6 expression in HDFs undergoing oxidative stress was inhibited only by HA-iMSC-EVs. Consistently, the dermal expression of α-SMA protein was reduced only by HA-iMSC-EVs. Previous study showed that IL-6 augments α-SMA expression and the differentiation of fibroblasts to myofibroblasts, which contract to bring the edges of the wound closer [ 64 ]. For this reason, therapeutic IL-6 blockade (tocilizumab) is currently used for treating fibrotic disease, such as systemic sclerosis [ 65 ]. Further detailed examinations should be followed by to determine the relationship between IL-6 and α-SMA expression during wound contraction induced by HA-iMSC-EVs. Skin grafting is one of the standard measurements for severe (e.g., third-grade) or large burn injury [ 66 ]. Mostly, skin substitutes facilitate re-epithelialization underneath the skin substitute, some of which are composed of allogenic/xenogenic matrix with or without autologous or allogenic cells [ 67 ]. Skin substitutes can be also incorporated with collagen fiber, xenogenic ECM, growth factors, keratinocytes, fibroblasts etc., all of which contributes to wound repair and scar improvement [ 68 ]. Considering the pleiotropic function of stem cell exosomes (i.e., immune regulation, cell proliferation, ECM deposition), loading stem cell exosomes into scaffold or dermal graft may open a novel therapeutic strategy for burn injury and reducing scar formation [ 69 ]. Conclusion To conclude, our results indicate that HA treatment of iMSCs produce EVs with an enhanced potential for the recovery of skin tissue after burn injury possibly by stimulating skin cell proliferation, capillary regrowth, and ECM production while reducing IL-6 and α-SMA expression. Declarations Ethics approval All authors performed experiments and analyses involving human materials and data under the Declaration of Helsinki. All human cells were commercially available. The ethics statement of the human cells used in the study follows the policy of the suppliers. Ethical statement can be found on their website; STEMCELL Technologies (https://www.stemcell.com/ipsc-faq.html#donor) and ScienCell Research Laboratories (https://sciencellonline.com/technical-support/ethical-statement.html). All animal experiments were conducted in Asan Medical Center (from December 2018 to March 2019), where S Lee and S Kim had previously worked at before joining Brexogen Inc. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Asan Medical Center (Approval date: Nov. 29, 2018; Approval number: 2018-12-284; Project title: Skin regeneration effects of human mesenchymal stem cell derived extracellular vesicles). Consent for publication Not applicable. Availability of data and materials The proteomics data of HA-iMSC-EVs analyzed in this study are provided as supplementary data (additional file 2: Table S2). Data and materials can be provided to the corresponding author via email upon request. Competing interests S Kim is the chief executive officer of Brexogen Inc. Other authors declare that they have no competing interests. Funding The research was supported with the research and development budget of Brexogen Inc. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT)(RS-2024-00336067). Author contributions M Jung and S Kim designed the experiments. M Jung, S Lee, EA Kim and H You performed the experiments. M Jung and HG Oh contributed to data analysis and interpretation. M Jung assembled the data and created the schematic and graphics. The manuscript was initially drafted by M Jung. TM Kim and S Kim supervised the study and wrote the manuscript. All authors gave final approval for the submitted version of the manuscript. Acknowledgements The authors declare that they have not used Artificial Intelligence in this study. Authors’ information 1 Brexogen Research Center, Brexogen Inc., Songpa-gu, Seoul 05855, South Korea. 2 Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang, Gangwon-do 25354, South Korea. 3 Institutes of Green-Bio Science and Technology, Seoul National University, Pyeongchang, Gangwon-do 25354, South Korea. References Jeschke MG, van Baar ME, Choudhry MA, Chung KK, Gibran NS, Logsetty S. Burn injury. Nat Reviews Disease Primers. 2020;6(1):11. 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Supplementary Files Additionalfile1R2.docx Additionalfile2TableS2R2.xls ArriveAuthorChecklistBurnwoundhealing.pdf 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4821606","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":348023208,"identity":"2e8d45e7-410e-4d37-ad25-ab0cd24aaddf","order_by":0,"name":"Minyoung Jung","email":"","orcid":"","institution":"R\u0026D Center, Brexogen Inc.","correspondingAuthor":false,"prefix":"","firstName":"Minyoung","middleName":"","lastName":"Jung","suffix":""},{"id":348023209,"identity":"3dcc2c23-a777-4817-b59c-ceaf9ac56288","order_by":1,"name":"Hyun Geun Oh","email":"","orcid":"","institution":"R\u0026D Center, Brexogen Inc.","correspondingAuthor":false,"prefix":"","firstName":"Hyun","middleName":"Geun","lastName":"Oh","suffix":""},{"id":348023210,"identity":"d0aa9876-e9b9-448e-9ce0-0bab35acca32","order_by":2,"name":"Seul Ki Lee","email":"","orcid":"","institution":"R\u0026D Center, Brexogen Inc.","correspondingAuthor":false,"prefix":"","firstName":"Seul","middleName":"Ki","lastName":"Lee","suffix":""},{"id":348023211,"identity":"92a2591b-fac9-49d3-b50a-c4f5a3760ef8","order_by":3,"name":"Eun A Kim","email":"","orcid":"","institution":"R\u0026D Center, Brexogen Inc.","correspondingAuthor":false,"prefix":"","firstName":"Eun","middleName":"A","lastName":"Kim","suffix":""},{"id":348023212,"identity":"cd6367fe-434d-4eca-b575-40bbc9cde443","order_by":4,"name":"Haedeun You","email":"","orcid":"","institution":"R\u0026D Center, Brexogen Inc.","correspondingAuthor":false,"prefix":"","firstName":"Haedeun","middleName":"","lastName":"You","suffix":""},{"id":348023213,"identity":"0d50ff19-c533-4614-b964-29a056ce3301","order_by":5,"name":"Tae Min Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYLCCBAYbHn4YR4KQah6IljQZyQaStDAwHLYxOECsFnv2w0c3PNzBzGN8/uwxCYYaOwbJ2Qfwa+HhSUu7kXiGjcfsRl6aBMOxZAZpvgRCDssxu5HYxgPUwmMmwcB2gEGOh4DDePjffwNqkeAx7j8D1PKPGC0SOWxALQY8BkDrJBjbDjBIE9Ry4xnIYQk8EjdyjC0S+5J5JHsIaGHvT35282fbf3v+/jOGNz58s5OTOENACypIgEfUKBgFo2AUjAKKAAD5mzkGbWLSLQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0015-2701","institution":"Seoul National University","correspondingAuthor":true,"prefix":"","firstName":"Tae","middleName":"Min","lastName":"Kim","suffix":""},{"id":348023214,"identity":"46f38fa6-3e0a-4419-90ec-0b69101765b9","order_by":6,"name":"Soo Kim","email":"","orcid":"","institution":"Brexogen Inc.","correspondingAuthor":false,"prefix":"","firstName":"Soo","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2024-07-29 11:25:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4821606/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4821606/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65722602,"identity":"f4e72795-3b97-42ff-a9c2-1e82af877918","added_by":"auto","created_at":"2024-10-01 17:14:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":112545,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of iMSC-EVs and HA-iMSC-EVs.\u003c/strong\u003e (A) HA-iMSC-EVs were measured the average size is 163.5 nm via NTA. (B) Immunoblotting analysis of HA-iMSCs and HA-iMSC-EVs for markers of extracellular vesicles. Uncropped western blot images are shown in Additional file 1: Supplementary Figure 4. (C) Flow cytometric analysis of iMSC-EVs and HA-iMSC-EVs.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-4821606/v1/9075ce695dd919cbd80a4e03.png"},{"id":65722601,"identity":"d0223b9b-16a0-4ba8-824c-f95f50e6c13b","added_by":"auto","created_at":"2024-10-01 17:14:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":213914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein set ontology and PPI analysis of proteins increased in HA-iMSC-EVs compared to iMSC-EVs. \u003c/strong\u003e(A) Bar charts of GO analysis using DAVID. GO terms were set with a cut-off at FDR \u0026lt; 0.05. (B) PPI network of cluster 1 from HA-iMSC-EVs.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-4821606/v1/28d96152f176d8dab2265855.png"},{"id":65723023,"identity":"05098ceb-2093-431b-9469-55efb64a3755","added_by":"auto","created_at":"2024-10-01 17:22:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":114377,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHA-iMSC-EVs increased cell viability and migration activity decreased by hydrogen peroxide.\u003c/strong\u003e (A) Cell viability according to hydrogen peroxide exposure in HDFs. Cell viability of HDFs exposed at H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations above 0.63 mM was decreased. (B) Restoration of cell viability according to treatment of HA-iMSC-EVs or control substrates. HA-iMSC-EVs increased cell viability in HDFs exposed to hydrogen peroxide. (C) Cell migration assay images. HA-iMSC-EVs increased cell migration activity in HDFs exposed to hydrogen peroxide. (D) A graph of cell migration assay. Data are expressed mean ± SEM. #, p-value \u0026lt; 0.05; ##, p-value \u0026lt; 0.01; ###, p-value \u0026lt; 0.001 versus PBS group without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exposure. *, p-value \u0026lt; 0.05; **, p-value \u0026lt; 0.01; ***, p-value \u0026lt; 0.001 versus PBS group with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exposure.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-4821606/v1/df8349b0f92d24cb1c2761bb.png"},{"id":65722610,"identity":"03778632-e6bd-4908-bce3-7966dc7798a2","added_by":"auto","created_at":"2024-10-01 17:14:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHA-iMSC-EVs increased growth factors in HDFs after H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e exposure\u003c/strong\u003e. (A) Graphs indicate the mRNA expression of pro-inflammatory cytokines, including TNF-α, IL-1b, and IL-6. (B) Graphs of the mRNA expression of growth factors, including TGF-β1, VEGF, IGF1, EGF, and HGF, respectively. Data are expressed mean ± SEM. #, p-value \u0026lt; 0.05; ##, p-value \u0026lt; 0.01; ###, p-value \u0026lt; 0.001 versus PBS group without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exposure. *, p-value \u0026lt; 0.05; **, p-value \u0026lt; 0.01; ***, p-value \u0026lt; 0.001 versus PBS group with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exposure.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-4821606/v1/7d08036ed2ad28b8497ff4e5.png"},{"id":65723024,"identity":"67bce74c-481e-4737-9cf2-a2b08e509f3f","added_by":"auto","created_at":"2024-10-01 17:22:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":209784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHA-iMSC-EVs restored ECM components.\u003c/strong\u003e (A) Type 1 collagen expression in ICC staining. Hydrogen peroxide was decreased type 1 collagen in HDFs, and HA-iMSC-EVs was increased type 1 collagen expression in HDF exposed to hydrogen peroxide. (B) Elastin expression in ICC staining. Hydrogen peroxide decreased elastin expression in HDFs, and HA-iMSC-EVs restored elastin expression in HDFs exposed to hydrogen peroxide. (C) A graph of type 1 collagen expression. (D) A graph of elastin expression in ICC staining. Data are expressed mean ± SEM. #, p-value \u0026lt; 0.05; ##, p-value \u0026lt; 0.01; ###, p-value \u0026lt; 0.001 versus PBS group without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exposure. *, p-value \u0026lt; 0.05; **, p-value \u0026lt; 0.01; ***, p-value \u0026lt; 0.001 versus PBS group with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exposure.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-4821606/v1/b90d57f83ac4c1ea130b65d3.png"},{"id":65724417,"identity":"8d2bbb84-642a-42f7-9817-bf4ca88b4315","added_by":"auto","created_at":"2024-10-01 17:38:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":357459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHA-iMSC-EVs accelerated wound closure.\u003c/strong\u003e (A) Progress of wound closure. On the Day 9, wound size of HA-iMSC-EVs group were decreased compared with PBS group. (B) Wound closure rate by date (n=5 for each group). Data are expressed mean ± SEM. *, p-value \u0026lt; 0.05; **, p-value \u0026lt; 0.01; ***, p-value \u0026lt; 0.001 versus PBS group.\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-4821606/v1/0928c01130c829247c6149b8.png"},{"id":65722605,"identity":"8eefb467-1953-4827-8fec-ccf028fe1e38","added_by":"auto","created_at":"2024-10-01 17:14:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":722777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHA-iMSC-EVs reconstructed histological changes in cutaneous burn wounds. \u003c/strong\u003e(A) Verhoeff van Gieson stain images. HA-iMSC-EVs increased dermal collagen and elastin compared PBS. (B) A graph of collagen density (n=5 for each group), (C) A graph of elastin density (n=5 for each group). These graph were revealed Verhoeff van Gieson stain images. (D) CD31 expressed micro vessels in burn wound lesions. Red dots and numbers are vessel counts. The vessels number of HA-iMSC-EVs groups were decrease to a basal levels, similar to the number of vessels of a non-lesion in PBS group. (E) A graph of micro vessel density (n=5 for each group). Data are expressed mean ± SEM. *, p-value \u0026lt; 0.05; **, p-value \u0026lt; 0.01; ***, p-value \u0026lt; 0.001 versus PBS group.\u003c/p\u003e","description":"","filename":"Slide7.png","url":"https://assets-eu.researchsquare.com/files/rs-4821606/v1/6921ddcc6b09163062da1898.png"},{"id":65723029,"identity":"361d90f1-9a5b-40ac-bfe9-397995e1530e","added_by":"auto","created_at":"2024-10-01 17:22:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":267084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHA-iMSC-EVs restored α-SMA expression.\u003c/strong\u003e (A) α-SMA expression in burn wound lesions. α-SMA is expressed during the early stages of the normal wound healing process. However, an abnormal increase of α-SMA in the late stages of wound healing is a sign of wound contraction. HA-iMSC-EVs decreased α-SMA expression, similar to a pattern of non-lesions in the PBS group. (B) A graph of α-SMA expression (n=5 for each group). Data are expressed mean ± SEM. *, p-value \u0026lt; 0.05; **, p-value \u0026lt; 0.01; ***, p-value \u0026lt; 0.001 versus PBS group.\u003c/p\u003e","description":"","filename":"Slide8.png","url":"https://assets-eu.researchsquare.com/files/rs-4821606/v1/05e47077ac071daf095d96e8.png"},{"id":70508054,"identity":"3e94418f-2df4-4e1c-b0d0-d476abd347ef","added_by":"auto","created_at":"2024-12-04 00:01:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2812656,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4821606/v1/e344db83-d442-41f5-a369-e003ecd1ab01.pdf"},{"id":65723028,"identity":"db608109-f1fe-42e1-a793-5c8dbcda70b5","added_by":"auto","created_at":"2024-10-01 17:22:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":823107,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1R2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4821606/v1/c942dd6a7a7c630ccf9abffd.docx"},{"id":65723907,"identity":"1eefd1d9-bb82-45ed-a0ce-f31f60ec6f25","added_by":"auto","created_at":"2024-10-01 17:30:48","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":49152,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2TableS2R2.xls","url":"https://assets-eu.researchsquare.com/files/rs-4821606/v1/86035a3f1624efd16c6999f7.xls"},{"id":65723025,"identity":"1924d672-8dd7-45fd-9fb1-4313a3e388b4","added_by":"auto","created_at":"2024-10-01 17:22:48","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":115563,"visible":true,"origin":"","legend":"","description":"","filename":"ArriveAuthorChecklistBurnwoundhealing.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4821606/v1/313f6a4742e8fffacbab9b6d.pdf"}],"financialInterests":"","formattedTitle":"Hyaluronic acid stimulation of induced MSCs produces extracellular vesicles with enhanced healing for skin burn wounds","fulltext":[{"header":"Background","content":"\u003cp\u003eBurn injuries remains a serious public health issue with a high prevalence and fatality rate. According to WHO estimates, 180,000 of the 11\u0026nbsp;million burn injuries occur annually worldwide are fatal [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The breakage of skin integrity provokes infection and compromised skin function, thus immediate treatment is needed. In severe burn injury, skin regrowth starts from marginal area, which makes the chance of infection high. The pathological feature from this injury include metabolic changes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], immune responses [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], which can lead to shock and multiple organ failure [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Moreover, the overall quality of life and psychological health can be profoundly impacted by burn injuries [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The current gold standard for managing burn wounds that are extensive or severe is autologous skin grafting followed by early excision [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Despite advances in technologies, managing and treating burn wounds still remains significant obstacles. polymorphonuclear neutrophils (PMNs) immediately invade the lesion and release large amounts of reactive oxygen species (ROS) that cause endothelial cell and skin damage. ROS increases capillary permeability and promote edema formation in the zone of stasis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The overproduction of free radicals also impairs the production and release of growth factors such as EGF, TGF-β, and VEGF, causing delayed wound healing [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thus, growth factors have been given attention as the key molecules regulating wound repair and regeneration by their therapeutic role in the pathophysiology of chronic wounds [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, their translation to the clinic has been limited because they have a short in vivo half-life due to their low stability and restricted absorption rate [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegenerative wound healing uses emerging biomedical research technologies, including targeted drug/growth factor delivery, smart wound dressings, bioactive biomaterial matrices, gene therapy, and stem cell therapy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The ideal healing of skin wounds involves regenerating damaged skin tissue while preventing the progression of chronic wounds and reducing scar formation (hypertrophic scar or keloid). Although the enormous impact of chronic wounds and fibrosis on human health and increasing prevalence and cost, chronic wounds and scar formation remain an unmet treatment area [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe feasibility of stem cell-based treatments for burn injury have been shown in recent preclinical studies, and this field is one of the most active areas that are under clinical investigation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In particular, many preclinical studies reported that various types of MSCs reduced fibrosis and repaired wounds [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It was also shown that MSCs promote wound healing via their paracrine function rather than their ability to differentiate into skin cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the therapeutic outcome of MSCs is not optimal, mostly due to multiple obstacles such as low survival in vivo, thrombosis, and tumorigenesis. Additionally, the therapeutic potential of MSCs reduces and they undergo cellular senescence during long term culture [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExtracellular vesicles (EVs) are lipid bilayered-particles that are essential for cell-to-cell communication because they carry certain biomolecular cargos that are necessary for tissue homeostasis and recovery. Importantly, EVs derived from MSCs (MSC-EVs) have several advantages over MSCs, including abundant resources, low immunological rejection, and the ability to be reengineered or combined with other biomaterials [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Moreover, different strategies (e.g., genetic modification or cell priming) can be implemented to modify the biomolecular cargo of EVs depending on the purpose [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Compared to other approaches, small molecule preconditioning/priming strategies have several advantages, such as being quick and simple to use and having well-established underlying processes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHyaluronic acid (HA) is an essential component of the ECM that has numerous functions including extracellular matrix function, cell migration, and wound repair [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this study, we assessed whether EVs from HA-primed iMSCs (HA-iMSC-EVs) can accelerate the recovery of burn injury in mice skin. Relevant \u003cem\u003ein vitro\u003c/em\u003e experiments were also conducted to determine the mechanism of their function.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEV isolation\u003c/h2\u003e \u003cp\u003eFor EV isolation, iMSCs were generated from induced pluripotent stem cells (STEMCELL Technologies Inc., Vancouver, Canada) as described in our previous study [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and iMSCs were established as a working cell bank at the R\u0026amp;D center of Brexogen Inc. (Seoul, Rep. of Korea). Briefly, established iMSCs were cultured in high-glucose Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; HyClone, Chicago, IL, USA) supplemented with 15% fetal bovine serum (FBS; HyClone) and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific, Waltham, MA, USA) at 37\u0026deg;C in a humidified incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e. At 90% confluence, cells were detached using TrypLE Express (Thermo Fisher Scientific) and seeded at a density of 10,000 cells/cm\u003csup\u003e2\u003c/sup\u003e. The next day, the cells were stimulated with or without 40 \u0026micro;g/mL HA (Sigma-Aldrich, St Louis, MO, USA) for 24 h. After being washed with Dulbecco\u0026rsquo;s Phosphate Buffered Saline (HyClone), cells were cultured in phenol red-free DMEM (Gibco, Waltham, MA, USA) supplemented with 15% EV-depleted FBS. After 3 d of incubation, the culture medium was harvested and centrifuged at 300 \u0026times; g for 5 min and 2,000 \u0026times; g for 30 min to remove cells and debris. The supernatant containing HA-iMSC-EVs was filtered using a vacuum filter (Merck, Darmstadt, Germany) and then was diafiltered with Dulbecco's Phosphate-Buffered Saline (DPBS, Gibco) by tangential flow filtration (TFF) systems (Sartorius AG, G\u0026ouml;ttingen, Germany) (Additional file 1: Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, A).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eNanoparticle tracking analysis\u003c/h2\u003e \u003cp\u003eThe particle size and concentration of EVs were measured Zetaview\u0026reg;BASIC NTA-Nanoparticle Tracking (Particle Metrix, Inning am Ammersee, Germany). The values for the standard controls were set as follows; Sensitivity: 80, Frame Rate: 30, Shutter: 100, Temperature: 23\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eTo characterize surface proteins of EVs, western blotting was performed as previously described [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Briefly, EVs were lysed in RIPA lysis buffer with protease inhibitors (Thermo Fisher Scientific). Protein concentration was measured using the Bradford Assay Reagent (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s protocol. Proteins were loaded and separated on precast polyacrylamide Mini-PROTEAN TGX gels (Bio-Rad Laboratories, Hercules, USA) and transferred to PVDF membranes (Bio-Rad Laboratories). The membranes were incubated overnight with primary antibodies at 4\u0026deg;C. Antibodies against CD63 (Abcam, Cambridge, UK) and TSG101 (Invitrogen, Waltham, MA, USA) were used as the primary antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003e HA-iMSC-EVs analysis was performed using human MACSPlex Exosome Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer\u0026rsquo;s instructions. Flow cytometric analysis was conducted using an Attune NxT flow cytometer (Thermo Fisher Scientific), as previously described [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eProteome profiling and bioinformatic proteome analyses\u003c/h2\u003e \u003cp\u003eTo compare protein sets of iMSC-EVs and HA-iMSC-EVs, liquid chromatography with tandem mass spectrometry (LC/MS\u0026ndash;MS) analysis was employed in CellKey Co., Ltd (Seoul, Rep. of Korea). Briefly, before LC/MS-MS analysis, proteins are extracted from each three EV batches using RIPA lusis buffer (Thermo Fisher Scientific). After protein digestion by trypsin, prepare the desalting peptide fraction for LC/MS-MS. An EASY-Spray PepMap Neo C18 column (Thermo Fisher Scientific) was used with the mobile phase consisting of buffer A (0.1% formic acid in water) and buffer B (0.1% formic acid in acetonitrile). A portion was injected at 300 nL/min into the LC system. MS\u0026ndash;MS analysis was carried out on a Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer in the nano ESI positive ion mode. Tryptic peptides were identified and quantified using Proteome Discoverer (Thermo Fisher Scientific). A database including 20,307 proteins from UniProt was used to identify proteins in the samples. The identification of proteins was performed with 1.0% of false discovery rate (FDR). Normalized peptide abundances were used for the label-free quantification of proteins. The fold change (Log\u003csub\u003e2\u003c/sub\u003eFC) of protein abundance values cutoff was set at 1.0 (two-fold up-regulation, p-adj\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Additional file 2: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProtein set ontology analysis was performed using the DAVID at an FDR q-value cutoff of 0.05 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The protein sets subjected to the Markov Cluster Algorithm (MCL) cluster analysis and protein-protein interaction (PPI) network with confidence of 0.7 using STRING 12.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHydrogen peroxide treatment and cell viability measurement using MTS assay\u003c/h2\u003e \u003cp\u003eHuman dermal fibroblasts (HDFs; ScienCell Research Laboratories, Carlsbad, CA, USA) were maintained in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM; GIBCO\u0026reg; Invitrogen, Carlsbad, CA, USA) containing penicillin (100 IU/ml), streptomycin (100 \u0026micro;g/ml), and 10% fetal bovine serum (FBS; GIBCO\u0026reg;) at 37℃ in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. HDFs at passages 8\u0026ndash;10 were manipulated for in-vitro assay. Cells were cultured in 96-well plates (5,000 cells/well) and grown overnight at 37˚C. Cells were incubated with various concentrations (0.16-5 mmol/L) of hydrogen peroxide for 3 hours at 37˚C under 5% CO\u003csub\u003e2\u003c/sub\u003e. Following exposure to mechanical strain and hydrogen peroxide, media was exchanged with the serum-free condition and treated with HA-iMSC-EVs at 0, 5, 10, 20, 40, 80 \u0026micro;g/ml. After 24 hours of incubation, MTS solution (CellTiter kit; Promega, Madison, WI, USA) was added to each well and incubated for one hour. The optical density at a wavelength of 490 nm was detected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell migration assay\u003c/h2\u003e \u003cp\u003eCell migration assay was conducted using a wound healing assay Kit (Abcam) according to the manufactured protocol. HDFs were seeded in 12-well plates (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well) assembled with inserts to make the gap (empty area) and 24 hours of cultivation, the inserts were removed, then cells were rinsed with DPBS. Following exposure to hydrogen peroxide for 3 hours, each plate added HA-iMSC-EVs or control vehicles with the DMEM media including 5% exosome depleted FBS, and cultivated them under standard cell culture conditions. At 0 h and 30 h, the wound healing was recorded by a microscope (Leica, Germany). Image J software was used to quantitatively evaluate the wound healing rate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative RT-PCR\u003c/h2\u003e \u003cp\u003eCells were harvested for total RNA extraction using a TRIzol\u0026reg; (Invitrogen) according to the manual protocol. Complementary DNA (cDNA) was then synthesized from 500 ng total RNA per sample using an AccuPower\u0026reg;RT PreMix kit (Bioneer Inc. Seoul, Rep. of Korea), followed by qPCR using a Power SYBR Green PCR Master Mix kit (Applied Biosystems). The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as an internal control and the qPCR primers used are listed in additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunocytochemistry\u003c/h2\u003e \u003cp\u003eThe cultured cells in 12-well plates were washed with DPBS and fixed in cold 100% methanol at -20℃. The cultures were incubated with 10 mg/ml of a polyclonal antibody to tropoelastin (Elastin Products, Owensville, MI) or polyclonal antibody to collagen type I (Abcam). Cultures were then incubated with the respective fluorescein-conjugated secondary antibodies. Nuclei were counter stained with DAPI (Invitrogen, Waltham, MA, USA). The stained cells were observed under a Nikon Eclipse Ti2-U fluorescent microscope (Nikon, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted in compliance with the approved animal protocols by IACUC of Asan Medical Center, Seoul, Korea (Approval No. 2018-12-284). 12-week-old female BALB/c mice (20-25g) were assigned randomly into three groups as follows: PBS group (n\u0026thinsp;=\u0026thinsp;5), iMSC-EVs group (n\u0026thinsp;=\u0026thinsp;5), and HA-iMSC-EVs group (n\u0026thinsp;=\u0026thinsp;5). The animals were kept under 25\u0026thinsp;\u0026plusmn;\u0026thinsp;3 ℃ temperature and relative humidity of 30\u0026ndash;70% in 12 h light/dark cycle with standard food and water were available \u003cem\u003ead libitum\u003c/em\u003e. The thermal injury model was developed by modifying the method of Tavares Pereira et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Briefly, fifteen mice were anesthetized with Zoletil (30 mg/kg, Virbac, Carros, France) and Rompun (10 mg/kg, Bayer, Leverkussen, Germany). Following anesthesia, the dorsal areas of the mice were shaved. An aluminum plate (diameter\u0026thinsp;=\u0026thinsp;10 mm) preheated at 100\u0026deg;C for 15 seconds was placed directly onto the depilated area to create symmetrical burns. Following the burn injury, burn wounds were covered with hydrocolloid dressing (DuoDerm\u0026reg;, ConvaTec, Berkshire, UK), then 100 \u0026micro;L HA-iMSC-EVs (1 mg/mL), iMSC-EVs (1 mg/mL), or PBS was used subcutaneously administrated. To minimize the variance that can arise from the order of treatments, administration was performed in following order: 1) iMSC-EVs, 2) HA-iMSC-EVs, and 3) PBS. The closure of burn wound was examined for 15 days (Additional file 1: Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, B). The sample size was estimated using the online Sample size calculator program v1.061 for ANOVA analysis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://homepage.univie.ac.at/robin.ristl/samplesize.php\u003c/span\u003e\u003cspan address=\"https://homepage.univie.ac.at/robin.ristl/samplesize.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) based on the wound closure rates from the pilot study (alpha\u0026thinsp;=\u0026thinsp;0.05, power\u0026thinsp;=\u0026thinsp;0.8). The study was reported in line with the ARRIVE guidelines 2.0, with additional supporting documents provided in the supplementary materials.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eThe mice were euthanized by CO\u003csub\u003e2\u003c/sub\u003e inhalation and the skin tissues were harvested at day 15 after thermal injury for burn wounds. Skin specimens were fixed in 4% paraformaldehyde, and paraffin embedded. H\u0026amp;E staining and Verhoeff van Gieson staining were performed according to routine protocols. And whole slides were scanned using Panoramic digital slide scanner (3DHISTECH), generating a TIFF file. The scanned images were evaluated blindly by two investigators. The evaluation range was limited to the dermis at equal distances from the basal layer of the epidermis in the wounded lesion. The region of interest (ROI) was set to the same size for each slide image at 200x magnification, five ROI values were measured for each slide, and the average value was calculated. The density of elastin and collagen fibers of each ROI was measured with Image J software program.\u003c/p\u003e \u003cp\u003eImmunohistochemical staining was performed using a primary antibody against mouse CD31 (Abcam, Cambridge, UK), or mouse alpha smooth muscle actin (α-SMA, Abcam). And histological images were conversed by whole slide scanner. The α-SMA density was quantified using Image J software program.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS (version 18.0 for IBM, Chicago, IL, USA). For comparisons involving three or more groups, one-way analysis of variance (ANOVA) was performed followed by Tukey\u0026rsquo;s post hoc test. Data are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SEM), and values with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of HA-iMSC-EVs\u003c/h2\u003e \u003cp\u003eTo confirm the characteristics of the iMSC-EVs and HA-iMSC-EVs, we measured the size of the EVs using NTA. The average diameters of iMSC-EVs and HA-iMSC-EVs were 130.4 and 136.9 nm, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Both the EV types expressed CD63 and TSG101 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Flow cytometry revealed that iMSC-EVs and HA-iMSC-EVs tested positive for CD9, CD63, and CD81, which are typical extracellular vesicle surface markers, but negative for stage-specific embryonic antigen-4 (SSEA4) and CD45 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results indicate that HA-iMSC-EVs met the general characteristics of EVs derived from cultured cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThe HA-iMSC-EVs proteome is associated with ECM construction-related functions\u003c/h2\u003e \u003cp\u003eTo determine the effect of HA preconditioning on the cargo protein profile, proteomic analysis was conducted by LC/LC-MS. A total of 821 proteins were identified in iMSC-EVs and HA-iMSC-EVs (Additional file 1: Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Among the 579 proteins identified in HA-iMSC-EVs, 422 proteins (72.9%) overlapped with those in iMSC-EVs. GO and KEGG analyses were performed on the protein sets that showed more than a 2-fold increase in HA-iMSC-EVs compared to those from iMSC-EVs from these overlapping genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). A total one hundred and fifty-nine proteins increased in HA-iMSC-EVs by HA preconditioning were analyzed using the DAVID program to identify the GO terms. The proteins set increased in HA-iMSC-EVs were related to the function of ECM, such as 'regulation of collagen fibril organization' (GO:190426, FDR\u0026thinsp;=\u0026thinsp;1.88E-02), 'extracellular matrix organization' (GO:0030198, FDR\u0026thinsp;=\u0026thinsp;1.91E-15) and 'collagen biosynthetic process' (GO:0032964, FDR\u0026thinsp;=\u0026thinsp;6.85E-03). Additionally, the protein set of HA-iMSC-EVs was related to ECM-receptor interaction (hsa04512, FDR\u0026thinsp;=\u0026thinsp;3.39E-07), tight junction (hsa045320, FDR\u0026thinsp;=\u0026thinsp;2.53E-03) and PI3K-AKT signaling (hsa04151, FDR\u0026thinsp;=\u0026thinsp;1.16E-04) in KEGG pathway analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThrough the STRING program, the proteins set was subjected to cluster analysis with Markov Cluster Algorithm. Then, the PPI network of the largest cluster containing the set of ECM-related proteins was analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB \u0026amp; Additional file 1: Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). As results, HA-iMSC-EVs were enriched with proteins related to collagen synthesis (FDR\u0026thinsp;=\u0026thinsp;7.66E-34) and elastic fiber formation (FDR\u0026thinsp;=\u0026thinsp;9.22E-12). KEGG pathway analysis showed that the proteins of HA-iMSC-EVs are involved in TGF-β (FDR\u0026thinsp;=\u0026thinsp;3.00E-03) and PI3K-AKT (FDR\u0026thinsp;=\u0026thinsp;2.59E-10) signaling pathways.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHA-iMSC-EVs enhance cell viability and migration of HDFs undergoing oxidative stress\u003c/h2\u003e \u003cp\u003eWe first examined the effect of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on the viability of HDFs by MTS assay. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the viability of HDFs was reduced under 0.63, 1.25, 2.5, and 50 mM of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). We used 0.63 mM of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for subsequent studies. HDFs were exposed to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 3 hours, and then incubated with serum-free medium containing iMSC-EVs or HA-iMSC-EVs for 24 hours. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, the viability was significantly higher in HDFs treated with 20 or 40 \u0026micro;g/mL HA-iMSC-EVs compared with those treated with PBS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In contrast, no change was observed in iMSC-EVs- or HA-treated cells. Consistently, HA-iMSC-EVs stimulated the migration of HDFs under oxidative stress compared with PBS-treated cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No effect was observed in iMSC-EVs- or HA-treated cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHA-iMSC-EVs decrease inflammatory cytokines expression and enhance growth factor expression in HDFs undergoing oxidative stress\u003c/h2\u003e \u003cp\u003eRT-qPCR analysis showed that hydrogen peroxide increased the mRNA expression of pro-inflammatory cytokines (i.e., \u003cem\u003eTNF-α, IL-1β\u003c/em\u003e, and \u003cem\u003eIL-6\u003c/em\u003e) in HDFs. HA-iMSC-EVs caused a slight reduction of the expression of \u003cem\u003eTNF-α\u003c/em\u003e and \u003cem\u003eIL-1β\u003c/em\u003e as compared with PBS. A significant decrease of \u003cem\u003eIL-6\u003c/em\u003e was found in HA-iMSC-EVs compared with PBS-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). No change in the mRNA expression of growth factors was observed after hydrogen peroxide treatment in HDFs. HA-iMSC-EVs augmented the mRNA expression of growth factors (\u003cem\u003eTGF-β1\u003c/em\u003e, \u003cem\u003eVEGF\u003c/em\u003e, \u003cem\u003eIGF1\u003c/em\u003e, \u003cem\u003eEGF\u003c/em\u003e, and \u003cem\u003eHGF\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No change was observed in iMSC-EVs- or HA-treated HDFs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eHA-iMSC-EVs increase type 1 collagen and elastin expression in HDFs undergoing oxidative stress\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, hydrogen peroxide decreased the protein expression of type I collagen and elastin. Both iMSC-EVs and HA-iMSC-EVs was able to augment the expression of Type 1 collagen (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and such increase was more significant in HA-iMSC-EVs compared with iMSC-EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) (p\u0026thinsp;=\u0026thinsp;0.048, t-test). Additionally, HA treatment led to an increase in type I collagen expression, which was comparable to the levels observed with iMSC-EVs (p\u0026thinsp;=\u0026thinsp;0.088, t-test). Similarly, both iMSC-EVs and HA-iMSC-EVs enhanced the protein expression of elastin in hydrogen peroxide-exposed HDFs. However, such increase was more significant in HA-iMSC-EVs compared with those from iMSC-EVs (p\u0026thinsp;=\u0026thinsp;0.015, t-test), which was comparable to those from intact cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). No increase in the expression elastin was noted in HA-treated cells. Interestingly, many elastin fibrils were observed in the HA-iMSC-EVs group, similar to those observed in intact cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In contrast, only accumulated tropoelastin was observed in the iMSC-EVs group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eHA-iMSC-EVs improves wound healing in burn injury of mice\u003c/h2\u003e \u003cp\u003eWe next evaluated whether HA-iMSC-EVs had a greater effect in the rate of wound closure after burn injury. In iMSC-EV, there was a minor increase in wound closure; however, no significant increase against PBS was found. In HA-iMSC-EVs-treated animals, however, the wound closure was significantly enhanced compared with those from animals receiving PBS. Even on Day 9, 80.9% of the burn wound was closed in the HA-iMSC-EVs group, which was higher than those found in the iMSC-EVs group on Day 13. On Day 15, no change was observed in the wound closure rate among three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eHA-iMSC-EVs enhance dermal matrix formation and reduce α-SMA\u003c/h2\u003e \u003cp\u003eMicroscopic analysis showed that both types of EVs enhanced the formation dermal matrix; however, such change was more prominent in HA-iMSC-EVs compared with iMSC-EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Quantitative measurement revealed that the increase in the positive area that reacted with collagen antibody was observed only in HA-iMSC-EVs-treated animals (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Both iMSC-EVs and HA-iMSC-EVs promoted the elastin density, however, such increase was more significant in HA-iMSC-EVs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 in iMSC-EVs and HA-iMSC-EVs, as compared with PBS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003eNeovascularization is essential for wound healing, as maladaptive vascularization is a common feature of chronic wounds [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Capillaries created during wound healing regress as granulation tissue converts into mature scar tissue. Eventually, the number of vessels returns to a level close to that observed in uninjured skin [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, a significant reduction in CD31-positive vessels was found only in HA-iMSC-EVs-treated mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. PBS); no effect was observed in iMSC-EVs.\u003c/p\u003e \u003cp\u003eThe expression of α-SMA is responsible for contraction of wound during early stage and eventually disappears [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, its persistent expression can be found in fibrotic scars [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The expression of α-SMA was decreased only in HA-iMSC-EVs as compared with PBS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, its diffuse expression was observed in iMSC-EVs, which was similar to those in PBS-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eExosomes control various biological processes, and they have showed potential in treating diseases including cardiovascular, immune, and neuronal diseases [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The horizontal transfer of biomolecules by exosomes was first demonstrated in 2007, whereby exosomal mRNA can be translated upon entering receiving cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Following this report, studies have demonstrated that exosomes carry lipids, proteins, microRNAs, lncRNAs, and circRNAs, which are involved in regulation of inflammatory response, cell proliferation, migration, angiogenesis, and ECM remodeling [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Importantly, these events are essential for successful wound healing, and maladaptive repair events can lead to chronic wound development.\u003c/p\u003e \u003cp\u003eThe aim of the present study was to examine whether the HA stimulation of iMSCs can produce EVs that have enhanced function in the recovery of skin burn injury. HA-iMSC-EVs had proteins associated with the maintenance of skin integrity (ECM production and organization, TGF-β signaling, and PI3-AKT signaling etc.). Under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress, the migration and viability of HDFs were increased by HA-iMSC-EVs compared with those treated with iMSC-EVs. The elevated level of IL-6 expression by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was reduced only by HA-iMSC-EVs. Additionally, the mRNA expression of key growth factors was increased only by HA-iMSC-EVs. Also, the decrease of elastin and collagen protein expression by oxidative stress was reversed by both iMSC-EVs and HA-iMSC-EVs, but its effect was more significant in HA-iMSC-EVs. In burn-injured mice, accelerated wound closure was observed only in animals that received HA-iMSC-EVs. The expression of collagen in skin tissue was enhanced only by HA-iMSC-EVs. Both iMSC-EVs and HA-iMSC-EVs stimulated elastin production, with the latter more significant. The recovery of capillary density as well as reduction of α-SMA expression in dermal layer was observed only in animals that received HA-iMSC-EVs. Together, these results suggest that HA treatment of iMSCs produce EVs having enhanced potential the recovery of skin tissue after burn injury by stimulating skin cell proliferation, growth factor production, vessel formation, and ECM production in the lesion.\u003c/p\u003e \u003cp\u003eSkin is the largest organ of the body and plays multiple roles including sensation, body heat regulation, protection, and host defense [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Skin is prone to various injuries including trauma, surgery, chronic disease, and burns. In healthy individuals, injured skin recovers through the four stages of wound healing: hemostasis, inflammation, proliferation, and remodeling [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. On the other hand, some chronic wounds in diabetes or ischemia can cause an impaired wound healing process characterized by hypoxia, oxygen radicals, matrix metalloproteases, granulation tissue formation, reduced angiogenesis and decreased collagen synthesis and organization [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Following an acute burn injury, a strong inflammatory response occurs, which is characterized by neutrophil and macrophage recruitment to the injury site [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The innate immune cells and injured tissue release a wide range of inflammatory cytokines and growth factors (e.g., IL-1, IL-6, TGF-β, EGF, VEGF). When the production and secretion of these cytokines become excessive, wound closure can be delayed or the injury can progress to chronic stage [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Previous studies showed that MSC-EVs can block inflammation of skin burn injury; Li et al. showed that exosomal miR-181c from human umbilical cord-derived MSCs inhibited inflammation by suppressing Toll-like receptor 4 (TLR4) signaling in skin burn injury model as well as those in LPS-stimulated macrophages [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. More recently, Liu et al. conducted single cell sequencing analysis from the peri-wound skin of mice that had undergone full-thickness excision injury, and found that exosomes from human umbilical cord MSCs led to an increase of the proportion of M2 macrophages and neutrophils [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Since inflammatory events can affect the degree of wound closure and scar formation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], it would be needed to elucidate whether HA-iMSC-EVs can also repress the inflammation by regulating macrophage polarization at the early phase after injury.\u003c/p\u003e \u003cp\u003eGrowth factors play crucial role in the wound healing process by regulating immune cells, promoting migration and proliferation of dermal cells (e.g., epithelial cells and fibroblasts), collagen synthesis, and differentiation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. For example, it was demonstrated that EGF is crucial for improvement of re-epithelialization [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], and that IGF-1 accelerates wound healing by promoting angiogenesis [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Also, HGF was shown to augment neovascularization, re-epithelialization of skin wounds, and granulation tissue formation [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. However, due to the complexity of molecular pathways and wound chronicity, the local application of a single exogenous growth factor is insufficient to improve burn wounds [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Therefore, delivering a combination of growth factors using methods with high diffusion efficiency and bioavailability into the burn lesions is important for wound healing. Previous study showed that HA-iMSC-EVs enhance human umbilical vein endothelial cells (HUVEC) tube formation and angiogenesis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our study demonstrated that HA-iMSC-EVs increased the expression of various growth factors in skin fibroblasts. Thus, it is likely that HA-iMSC-EVs accelerated wound healing by improving re-epithelialization and neovascularization.\u003c/p\u003e \u003cp\u003eECM deposition is the last phase during wound healing process, and its failure can lead to chronic wound or excessive scar formation. Collagen and elastin are the two most essential ECM proteins in skin [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], and collagen accounts for 50\u0026ndash;90% of the dermal matrix. In line with our findings, Kim et al. reported that human umbilical cord-derived MSCs enhanced the cell migration as well as the synthesis of collagen and elastin in HDFs [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Furthermore, it has been shown that exosomes produced from human-induced pluripotent stem cell-derived MSCs (hiPSC-MSCs) promoted the expression of elastin, collagen I, and collagen III in HDFs [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In contrast, other studies demonstrated that MSC-derived exosomes inhibit the excessive production of ECM as well as fibroblast-to-myofibroblast transition, preventing scar formation. For example, it was shown that ADSC-derived exosomes reduced excessive scar formation by keeping fibroblasts from developing into myofibroblasts as well as controlling the ratios of TGF-β3/TGF-β1, collagen III/collagen I, and MMP3/TIMP1 [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. We demonstrated that excessive expression of α-SMA was inhibited by HA-iMSC-EVs, which might have contributed to inhibiting excessive scar formation by the reducing the number of myofibroblasts, while increasing structural integrity (e.g., by collagen production) in dermis.\u003c/p\u003e \u003cp\u003eElastin is responsible for tensile strength, providing structural integrity of skin. Mature, functional elastin is assembled from tropoelastin monomers through coacervation, cross-linking, and deposition [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. However, the synthesis of new tropoelastin stops after the neonatal period [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], and insufficient elastic fiber network contributes to the reduced elasticity and resilience of the mature scar [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The restoration of an immediate and functional elastic fiber is, therefore, critical to regain complete skin function after injury, and it is needed to develop novel strategy to increase elastin to restore skin function. Our results demonstrated that the increase of elastin expression was higher in HA-iMSC-EVs compared with iMSC-EVs in the recovered skin. In the fibroblasts, elastin fibrils were observed only in the HA-iMSC-EVs. In support of this findings, bioinformatic analysis showed that HA-iMSC-EVs have proteomic profile that are related to ECM composition, and that enriched proteins in HA-iMSC-EVs were related to elastic fiber formation and collagen biosynthesis. One of the key protein families required for tissue remodeling after injury is matrix metalloproteinases (MMPs) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. It was shown that hADSC-Exos activated MAPK pathway, stimulating the production of MMP-3 and TIMP-1, which led to enhanced ECM remodeling [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Another study demonstrated that miRNA-21 in hADSC-Exos promoted MMP-9 levels, while decreased TIMP2 as well as TGF-β1, thus reducing the formation of wound scars [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Zhang et al. reported that hADSC-exos augmented MMP-1 expression and downregulated α-SMA expression, and that promoted collagen deposition, improving dermal thickening in full-thickness incision wound model [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Thus, other in-depth studies such as the role of HA-iMSC-EVs in regulating MMPs/TIMPs and myofibroblast activation are needed to further examine detailed mechanisms how ECM deposition was increased in dermal layer.\u003c/p\u003e \u003cp\u003eWe observed that the increase of IL-6 expression in HDFs undergoing oxidative stress was inhibited only by HA-iMSC-EVs. Consistently, the dermal expression of α-SMA protein was reduced only by HA-iMSC-EVs. Previous study showed that IL-6 augments α-SMA expression and the differentiation of fibroblasts to myofibroblasts, which contract to bring the edges of the wound closer [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. For this reason, therapeutic IL-6 blockade (tocilizumab) is currently used for treating fibrotic disease, such as systemic sclerosis [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Further detailed examinations should be followed by to determine the relationship between IL-6 and α-SMA expression during wound contraction induced by HA-iMSC-EVs.\u003c/p\u003e \u003cp\u003eSkin grafting is one of the standard measurements for severe (e.g., third-grade) or large burn injury [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Mostly, skin substitutes facilitate re-epithelialization underneath the skin substitute, some of which are composed of allogenic/xenogenic matrix with or without autologous or allogenic cells [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Skin substitutes can be also incorporated with collagen fiber, xenogenic ECM, growth factors, keratinocytes, fibroblasts etc., all of which contributes to wound repair and scar improvement [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Considering the pleiotropic function of stem cell exosomes (i.e., immune regulation, cell proliferation, ECM deposition), loading stem cell exosomes into scaffold or dermal graft may open a novel therapeutic strategy for burn injury and reducing scar formation [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo conclude, our results indicate that HA treatment of iMSCs produce EVs with an enhanced potential for the recovery of skin tissue after burn injury possibly by stimulating skin cell proliferation, capillary regrowth, and ECM production while reducing IL-6 and α-SMA expression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors performed experiments and analyses involving human materials and data under the Declaration of Helsinki. All human cells were commercially available. The ethics statement of the human cells used in the study follows the policy of the suppliers. Ethical statement can be found on their website; STEMCELL Technologies (https://www.stemcell.com/ipsc-faq.html#donor) and ScienCell Research Laboratories (https://sciencellonline.com/technical-support/ethical-statement.html). All animal experiments were conducted in Asan Medical Center (from December 2018 to March 2019), where S Lee and S Kim had previously worked at before joining Brexogen Inc. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Asan Medical Center (Approval date: Nov. 29, 2018; Approval number: 2018-12-284; Project title: Skin regeneration effects of human mesenchymal stem cell derived extracellular vesicles).\u0026nbsp;\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\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proteomics data of HA-iMSC-EVs analyzed in this study are provided as supplementary data (additional file 2: Table S2). Data and materials can be provided to the corresponding author via email upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS Kim is the chief executive officer of Brexogen Inc. Other authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported with the research and development budget of Brexogen Inc. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT)(RS-2024-00336067).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM Jung and S Kim designed the experiments. M Jung, S Lee, EA Kim and H You performed the experiments. M Jung and HG Oh contributed to data analysis and interpretation. M Jung assembled the data and created the schematic and graphics. The manuscript was initially drafted by M Jung. TM Kim and S Kim supervised the study and wrote the manuscript. All authors gave final approval for the submitted version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have not used Artificial Intelligence in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eBrexogen Research Center, Brexogen Inc., Songpa-gu, Seoul 05855, South Korea. \u003csup\u003e2\u003c/sup\u003eGraduate School of International Agricultural Technology, Seoul National University, Pyeongchang, Gangwon-do 25354, South Korea. \u003csup\u003e3\u003c/sup\u003eInstitutes of Green-Bio Science and Technology, Seoul National University, Pyeongchang, Gangwon-do 25354, South Korea.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJeschke MG, van Baar ME, Choudhry MA, Chung KK, Gibran NS, Logsetty S. Burn injury. 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Sci Rep. 2017;7:13321.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeo GC, Keeley FW, Weiss AS. Coacervation of tropoelastin. Adv Colloid Interface Sci. 2011;167:94\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchultz GS, Wysocki A. Interactions between extracellular matrix and growth factors in wound healing. Wound Repair Regen. 2009;17:153\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Vries HJ, Middelkoop E, Mekkes JR, Dutrieux RP, Wildevuur CH, Westerhof H. Dermal regeneration in native non-cross-linked collagen sponges with different extracellular matrix molecules. Wound Repair Regen. 1994;2:37\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeiliang Z, Lili G. Research Advances in the Application of Adipose-Derived Stem Cells Derived Exosomes in Cutaneous Wound Healing. 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Stem Cell Res Ther. 2022;13(1):24.\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":"Extracellular vesicles, burn wound, chronic wound, extracellular matrix, growth factors.","lastPublishedDoi":"10.21203/rs.3.rs-4821606/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4821606/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSkin injuries occur for various reasons during whole life. Some chronic wounds could cause an impaired wound healing process characterized by wound hypoxia, high levels of oxygen radicals, elevated levels of matrix metalloproteases, delayed cellular infiltration and granulation tissue formation, reduced angiogenesis, decreased collagen synthesis and organization. In this study, we report the EVs from hyaluronic acid-primed iMSCs (HA-iMSC-EVs) accelerating wound healing and regenerating damaged tissues by inducing the various growth factors in the thermal injury of mice.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eEVs were collected from iMSCs primed with HA (HA-iMSC-EVs) or without HA (iMSC-EVs) and were isolated using TFF systems. Both EVs analyzed the characteristics. We investigated the proteome of HA-iMSC-EVs using the protein set ontology analysis and protein-protein interaction network. To evaluate the effect of HA-iMSC-EVs on the oxidative stress-induced wound healing delayed model, we assessed the effect of EVs on cell viability, cell migration rate, and the mRNA expression of growth factors using a hydrogen peroxide-exposed HDF model. In addition, we observed elastin and collagen expressions using an ICC staining in the HDF model. In thermal burn wound mice (BALB/c), we compared the effect of EVs in wound closure rate and histological analysis, including expression of elastin, collagen, α-SMA, and CD31.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHA-iMSC-EVs exhibited typical EV characteristics, including size distribution, markers, and surface protein expression. In GO term analysis, HA-iMSC-EVs increased the proteins associated with ECM, including collagen biosynthesis and elastin fiber formation. In hydrogen peroxide exposed HDF models, HA-iMSC-EVs notably increased cell viability and migration activity. Furthermore, HA-iMSC-EVs increased RNA expression of \u003cem\u003eVEGF, IGF1\u003c/em\u003e, and \u003cem\u003eHGF\u003c/em\u003e and decreased \u003cem\u003eIL-6\u003c/em\u003e mRNA expression compared to the PBS group. Elastin and collagen expression in the HA-iMSC-EVs group were also significantly increased. In burn-injured mice, HA-iMSC-EVs accelerated wound closure and enhanced histological recovery. HA-iMSC-EVs increased collagen and elastin density on the upper dermis and decreased α-SMA expression. Additionally, HA-iMSC-EVs promoted the capillary density in the dermis.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur results suggest that HA-iMSC-EVs accelerated the recovery from burn wound by providing ECM composition signal and regulating growth factors. Our strategy may contribute to the development of alternative treatment option for burn wounds.\u003c/p\u003e\u003ch2\u003eTrial registration\u003c/h2\u003e \u003cp\u003e: Not applicable.\u003c/p\u003e","manuscriptTitle":"Hyaluronic acid stimulation of induced MSCs produces extracellular vesicles with enhanced healing for skin burn wounds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-01 17:14:43","doi":"10.21203/rs.3.rs-4821606/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":"2e94ae5a-832f-4a30-b1d0-e165d59c53d0","owner":[],"postedDate":"October 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-03T23:53:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-01 17:14:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4821606","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4821606","identity":"rs-4821606","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}