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

doi:10.21203/rs.3.rs-4821606/v1

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Research Article

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

https://doi.org/10.21203/rs.3.rs-4821606/v1

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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.

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.

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% CO₂. At 90% confluence, cells were detached using TrypLE Express (Thermo Fisher Scientific) and seeded at a density of 10,000 cells/cm². 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 (Log₂FC) 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% CO₂ 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% CO₂. 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 (5×10⁴ 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 CO₂ 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.

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. 1A). Both the EV types expressed CD63 and TSG101 (Fig. 1B). 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. 1C). 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. 2A). 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. 2B & 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 H₂O₂ on the viability of HDFs by MTS assay. As presented in Fig. 3A, the viability of HDFs was reduced under 0.63, 1.25, 2.5, and 50 mM of H₂O₂ (p < 0.001). We used 0.63 mM of H₂O₂ for subsequent studies. HDFs were exposed to H₂O₂ for 3 hours, and then incubated with serum-free medium containing iMSC-EVs or HA-iMSC-EVs for 24 hours. As shown in Fig. 3B, 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. 3C and 3D).

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. 4A). 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. 4B, 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. 5C) (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. 5D). 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. 5B). 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. 6A, 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. 7A). 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. 7B, 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. 7D and 7E, 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. 7A, B).

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 H₂O₂-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 H₂O₂ 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].

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.

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

¹Brexogen Research Center, Brexogen Inc., Songpa-gu, Seoul 05855, South Korea. ²Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang, Gangwon-do 25354, South Korea. ³Institutes of Green-Bio Science and Technology, Seoul National University, Pyeongchang, Gangwon-do 25354, South Korea.

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You are reading this latest preprint version

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

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Trial registration

Figures

Background

Materials and methods

EV isolation

Nanoparticle tracking analysis

Immunoblotting

Flow cytometry

Proteome profiling and bioinformatic proteome analyses

Hydrogen peroxide treatment and cell viability measurement using MTS assay

Cell migration assay

Quantitative RT-PCR

Immunocytochemistry

Animal experiments

Histological analysis

Statistical analyses

Results

Characterization of HA-iMSC-EVs

The HA-iMSC-EVs proteome is associated with ECM construction-related functions

HA-iMSC-EVs enhance cell viability and migration of HDFs undergoing oxidative stress

HA-iMSC-EVs decrease inflammatory cytokines expression and enhance growth factor expression in HDFs undergoing oxidative stress

HA-iMSC-EVs increase type 1 collagen and elastin expression in HDFs undergoing oxidative stress

HA-iMSC-EVs improves wound healing in burn injury of mice

HA-iMSC-EVs enhance dermal matrix formation and reduce α-SMA

Discussion

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1