Experimental study on the effect and mechanism of adipose stem cell‐derived exosomes combined with botulinum toxin A on skin trauma in rats

Adipose stem cell‐derived exosomes (ADSC‐EXO) and botulinum toxin type A (BTX‐A) individually showed a therapeutic effect on skin wound repair.


| INTRODUC TI ON
Skin wound healing is a complex process in tissue repair and remodeling, 1,2 which includes the occurrence of a mechanical, physical, and/or chemical wounds, tissue inflammation, cell proliferation, and remodeling.Alteration of these steps could result in different complications.The most common long-term complications include scar formation or a series of problems, like secondary infection, slow wound healing, and/or hair loss. 3To date, it lacks an international consensus on treating skin wound repair.Thus, research on skin wound healing and repair could help us better understand the molecular mechanisms and provide novel strategies for wound repair.
To this end, adipose stem cells (ADSC) possess multiple potent mesenchymal stem cell properties.They can promote wound healing through the paracrine action of various cytokines and growth factors to regulate the biological functions of fibroblasts and photoaging wound repair and scar formation. 4Paracrine, indirect linkage, and direct intercellular contact are three common ways to facilitate cell-to-cell interactions.Exosomes (EXO) plays a pivotal role in paracrine signaling and can escape the host's immune response.So it has attracted researchers to conduct basic and clinical research on them. 5,6EXO usually has a 30-180 nm vesicular structure in the cells or eukaryotic cell fluid.It contains bioactive molecules, like cytokines and growth factors, signaling lipids, RNA, and DNA, which can be transferred into its target cells. 7Another study reported that EXO could enhance the cell self-repair and regeneration in the damaged tissue to accelerate wound healing and recover tissue homeostasis. 80][11] However, the precise molecular mechanism by which ADSC-EXO affects wound healing needs investigation.-A) is an effective neurotoxin produced by the bacteria Clostridium botulinum and related species. 12It inhibits the release of the neurotransmitter acetylcholine from axon endings to block the transmission of signals from the neuromuscular junction, thereby inhibiting muscle contraction, which lasts for approximately 2-6 months. 13BTX-A has been used for different therapeutic or cosmetic purposes for the past 20 years, 14 for example, it can improve facial wrinkles and contour reduction of calf muscle. 15,16BTX-A was also reported to prevent muscle pulling, microtrauma, inflammation during wound healing, and reduction of scar formation. 17,18However, the effects of BTX-A on wound repair are less studied.This study investigated the effects of ADSC-EXO and BTX-A and their combination on wound healing, prevention of scar formation, and molecular signaling events using the in vitro and in vivo models.We expect to provide insightful information regarding their use in the treatment of wound repair.

| Animals
The animal protocol of this study was approved by the Institutional Animal Care and Use Committee (IACUC) of Hainan Medical University.Adult SF Sprague-Dawley (SD) rats at 7-8 weeks of age were purchased from Changsha Tianqin Company and housed in a specific pathogen-free (SPF) "barrier" facility under the controlled temperature and humidity and alternating 12-h light and dark cycles.

| ADSC isolation and culture
The fat in the groin area of SD rats was resected, rinsed in normal saline, dissected into pieces, and then digested in 1% collagenase (Servicebio) at 37°C for 1 h, centrifuged at 1300 rpm at the room temperature for 10 min, and suspended in serum and xeno-free medium (MesenCult™-XF; Stem cell Technologies) through centrifugation by repeating the procedure twice.The precipitates were filtered using a 70μm filter (Falcon), transferred to a six-well dish, and cultured in a humidified incubator with 5% CO 2 at 37°C for 24 h.After that, the ADSCs were passaged five times and used for experiments.

| Immunofluorescence
ADSCs were analyzed by immunofluorescence for CD29 expression.Mainly, ADSCs were grown in six-well dishes, then fixed with 4% paraformaldehyde solution for 10 min at room temperature, and washed with PBS.Next, the cells were incubated with an anti-CD29 antibody (Maker, city, state) at 4°C overnight.The next day, the cells were incubated with a secondary antibody conjugated with fluorescein isothiocyanate (FITC) at room temperature for 1 h.After that, the cells were reviewed and scored under an inverted microscope (Olympus).

Conclusions:
The ADSC-EXO plus BTX-A treatment demonstrated a synergistic effect on skin wound healing through upregulation of VEGF expression and the TGF-β3/TGF-β1 and COL III/COL I ratio.

K E Y W O R D S
adipose tissue-derived stem cells, botulinum toxin, exosomes, skin wound healing

| ADSC-EXO isolation and culture
ADSCs at passage 5 were cultured to reach 85% polymerization.The medium was then refreshed with a serum-free one, and ADSCs were continuously cultured for 72 h.After that, the culture media were collected for ultrafiltration and centrifugation at 300× g for 10 min at 4°C to remove any alive cells and collected the supernatant.The supernatants were further centrifuged at 2000× g for 10 min and at 10000× g for 30 min to remove any dead cells and cell debris.
The resulting supernatants were filtered using a 0.22μm filter and concentrated using the Macrosep ultrafiltration tube.Next, the supernatants were collected through centrifugation thrice at 5000× g for 30 min and then centrifuged at 100 000× g for 70 min at 4°C.The precipitates were collected and resuspended in Dulbecco's phosphate-buffered saline (DPBS).

| Trilineage differentiation assay
To assess the adipogenic differentiation, we seeded the cells into 12-well plates (1 × 10 5 cells/well) and cultured them in DMEM supplemented with 10% FBS and a 1% antibiotic-antimycotic solution until full confluency.We then changed cell culture medium to the Stem Cells Adipogenesis Differentiation one (Cyagen) twice weekly.After 20 days of culture, we analyzed the adipogenesis using the oil red O staining and scored the cells under a microscope (Olympus).
To assess the osteogenic differentiation, we seeded the cells into 12-well plates (1 × 10 5 cells/well) and cultured in DMEM supplemented with 10% FBS and a 1% antibiotic-antimycotic solution.The next day, the medium was changed to Stem Cells Osteogenesis Differentiation one (Cyagen) twice weekly.The osteogenesis was analyzed with Alizarin red staining after 20 days of cell culture.
We cultured the cells using the Chondrogenic Differentiation Basal Medium (BI, Israel) to detect the chondrogenic differentiation.
In brief, 1 × 10 5 cells suspended in 0.5 mL medium were placed into a 15 mL polypropylene tube and centrifuged at 300× g for 5 min.
Then, the tube cap was loosened, and the cells were incubated at 37°C with 5% CO 2 .The medium was changed every 3 days.After 20 days, the cell pellet was fixed in 10% formalin and embedded in paraffin.After that, the cell pellet was cut into 5μm-thick sections and stained with Alcian blue.

| Nanoparticle tracking assay of ADSC-EXO
We performed the Nanoparticle Tracking Analysis (NTA, Particle Metrix), an instrument used to detect the concentration and particle size of substances like exosomes.In brief, we configured the standards, calibrated the instrument according to the operating procedures, and then measured the ADSC-EXO samples accordingly.

| Transmission electron microscopy
The ADSC-EXO was prepared at a 1:5000 dilution and added onto the copper net with drops.We then replaced the copper nets under the baking lamp for semi-drying and stained them with 1% phosphotungstate acid for 70 s.After removing the dye solution using filter paper, we placed the copper nets onto filter paper, baked them under the backing lamp for 5 min, and then reviewed them under a transmission electron microscope (JEOL Ltd.) at 60 kV voltage.

| Human skin fibroblasts and culture
Human skin fibroblasts (HSF) were obtained from Biosharp Company (Cat.#341540).To culture them, the HSF in a cryo-storage tube were thawed in warm water, transferred to a centrifuge tube containing culture medium, centrifuged at 1350 rpm for 5 min, discarded the supernatant, and added with 2 mL Dulbecco's modified Eagle's medium (DMEM) with high glucose (DMEM-H; Biosharp).
After that, HSF were seeded into a T25 flask containing 4 mL complete culture medium (450 mL DMEM-H + 10% fetal bovine serum [Biosharp] + 5 mL p/s [Biosharp]) and cultured in a humidified incubator with 5% CO 2 at 37°C.When HSF reached 80% confluency, cells were subcultured using 0.25% trypsin (Biosharp), while HSF between passages 4 and 6 were used for our experiments.HSF were cultured overnight and then treated with or without 1 × 10 6 particles of ADSC-EXO, 0.6 mL Botulinum toxin type A (3 U/mL; Lanzhou Institute of Biological Products), and their combinations for up to 24 h and analyzed subsequently.

| Flow cytometry
We performed the flow cytometric analysis to assess CD34 expressions in HSF using the CD34 kit and the flow cytometer BD Accuri™ C6 (BD Biosciences).In brief, HSF after treatment were collected and subsequently stained with the FITC-conjugated antibodies against CD34 at a dilution of 1:100 (Cat.#11-0349; eBioscience) according to the manufacturer's protocol.The flow cytometric data were analyzed using the CFlow plus 1.0 software (BD Biosciences).

| In vitro tracking assay
We performed the in vitro tracking assay using a kit from Sigma Chemicals.In particular, we mixed 5 μL of the PKH67 staining solution (Sigma) into 0.5 mL of Diluent C and then resuspended the ADSC-EXO in 0.5 mL of Diluent C and added the mixture into ADSC-EXO.Next, the mixture was incubated at room temperature for 6 min and added a serum-free medium to stop the reaction.After centrifugation, the supernatant was removed, and the precipitate was suspended at 4°C for 70 min and then added into HSF culture for up to 24 h.HSF were then reviewed under an inverted microscope (Olympus) and photographed.

| HSF wound healing assay
HSF were seeded into six-well dishes at 3 × 10 5 cells/well and cultured in a serum-free medium for 24 h to reach 95% or confluency.
Afterward, a wound was created using a 1000μl spear tip across the dishes.The HSF cultures were randomly divided into Group A (negative control with PBS), Group B (ADSC-EXO treatment), Group C (3 U/mL BTX-A treatment), and Group D (ADSC-EXO plus BTX-A treatment).The cells were reviewed and photographed under an inverted microscope (Olympus) at 0, 12, and 24 h, respectively.The experiment was in duplicate and repeated at least three times.

| In vivo rat skin wound model and treatment
We first established a round wound with a 10 mm diameter on the back of SD rats and removed the whole skin layer of the wound area to construct the wound model.We then randomly divided the rats into Group PBS (100 μL each of PBS + 0.9% NaCl), Group EXO (100 μL each of ADSC-EXO with 2.5 × 10 7 particles +0.9% NaCl), Group BTX (100 μL each of PBS + 3 U /ml BTX-A), and Group EXO/BTX (100 μL each of EXO with 2.5 × 10 7 particles +3 U /ml BTX-A).Each group was injected with the indicated agents into the wound area, while the ADSC-EXO was injected under the skin at the wound area on the first, third, fifth, and seventh day after surgery.BTX-A was injected subcutaneously in 5 mm near the wound area immediately after surgery.Changes in the wound size were observed and measured on Days 1, 5, 9, and 13, respectively.

| Histological analysis
On Day 16 after surgery, wound site skin specimens were dissected, fixed in 10% formalin, and subsequently embedded in paraffin to prepare 5 μm-thick tissue sections.Next, the sections were stained with hematoxylin and eosin (H&E) and reviewed and quantified under a light microscope (Olympus) to regenerate epithelium, infiltrating cells, collagen fiber bundle, and angiogenesis.

| Western blot
The skin tissue samples were cut into 1 × 1 mm 3 pieces and placed into a homogenate tube.The tissue samples were digested in a mixture of the cracking buffer supplemented with protease inhibitors (Beyotime).After centrifugation, the supernatants were collected and quantified using the bicinchoninic acid (BCA) protein assay kit (CWBIO).An equal amount of each protein sample was separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels using electrophoresis and then transferred onto the nitrocellulose membranes (Pall).For Western blotting, the membranes were incubated in 5% skimmed milk powder solution for 1 h at room temperature and then with primary antibodies in the blocking buffer at 4°C overnight.The membranes were then washed with Tris-based saline-Tween 20 (TBS-T) and incubated with secondary

| Immunohistochemistry
The level of VEGFA protein was assessed in skin tissue specimens using immunohistochemistry.In brief, the formalin-fixed and paraffin-embedded skin tissue sections were deparaffinized in xylene and rehydrated in a series of ethanol solutions (100-50%) and into tape water.The sections in a citric buffer (pH 5.0) were subjected to the antigen repair in a microwave and then incubated in normal serum-PBS (1:20 dilution) at room temperature for 1 h and then with an anti-VEGFA antibody (Abcam) at 4°C overnight.The following day, the sections were washed with PC + BS thrice and further incubated with a secondary antibody (Abcam) at a dilution of 1:200 at room temperature for 1 h.After brief washing with PBS, the sections were incubated with a DAB kit (Solarbio) at room temperature in the dark for 10 min and then incubated with 3,3′-diaminobenzidine (DAB) solution and counterstained with hematoxylin and mounted with coverslips.The stained sections were then reviewed and scored under a light microscope (Olympus).

| Masson's trichrome staining
After dewaxed and rehydrated, the tissue sections were stained with 1% Ponceau S Red for 5 min and then subsequently incubated in 1% phosphomolybdic acid for 5 min, 2.5% aniline blue for 5 min, and 1% glacial acetic for 1 min.After being washed in tap water briefly, the sections were counterstained in Harris' hematoxylin for 10 min and then incubated in 1% hydrochloric acid alcohol for 5 min and washed in warm tap water.Next, the sections were dehydrated in 95%-100% ethanol, cleared in xylene, and sealed.The sections were reviewed and photographed under a light microscope (Model BX41; Olympus), and the images were analyzed using Image-Pro Plus software (US National Institutes of Health).

| Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)
Total RNA was isolated from the animal tissue samples using a Trizol reagent and then reversely transcribed into cDNA using a reverse transcription kit (Tiangen) according to the manufacturers' protocols.After that, the resulting cDNA samples were subjected to qPCR amplification of VEGF-A and GAPDH mRNA (the latter was used as an internal control).The thermal cycling conditions were set as followings, that is, 1 min at 95°C and then 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s.The reactions were performed in duplicate.The specific oligos used in qPCR were VEGF-A, 5'-CA ATG ATG A AG CCC TGG AGTG-3′ and 5'-GCTCA TCT CTC CTA TGT GCTGG-3′ and GAPDH, 5'-CTGGA GA A ACC TGC CA A GTATG-3′ and 5'-GGTGG A AG A AT GGG AGT TGCT-3′.Level of gene expression was calculated using the 2 -ΔΔCT relative expression method.

| Statistical analysis
The experiments were repeated at least thrice.The data were summarized as mean ± SEM when properly analyzed using SPSS 20.0 (SPSS).The Student's t-test was performed for the statistical analysis, and a p < 0.05 was considered statistically significant.

| Characterization of ADSC and ADSC-EXO
In this study, we first isolated ADSCs and ADSC-EXO from the primary culture of rat fat tissues.In vitro, ADSCs were in spindle shape and irregularly arranged (Figure 1A), and CD29-positive were analyzed by immunofluorescence (Figure 1B).The CD29-positive rate in ADSCs was 87.7% (Figure 1C) and CD45, and the 31-positive rate was 30.6% (Figure 1D) analyzed using flow cytometry.The ADSCs maintained their trilineage differentiation ability (Figure 1E-G).
After that, we isolated ADSC-EXO from the cultured ADSCs at passage 5 and assessed them using transmission electron microscopy, which showed them to be circular vesicles with a diameter of 30-180 nm (Figure 1H), and the peak size was 120 nm (Figure 1I), consistent with the general exosome morphological characteristics.After being diluted 1000 times with ddH 2 O, the median particle size of the ADSC-EXO was 145.9 nm, and the concentration

| Promotion of fibroblast migration and wound healing after ADSC-EXO plus BTX-A treatment in vitro and in vivo
Next, we labeled the ADSC-EXO with PKH67 to demonstrate whether the ADSC-EXO could be up-taken by HSF.Notably, we co-cultured HSF with ADSC-EXO for 2 h and found that ADSC-EXO could enter into HSF under a fluorescence microscope (Figure 2B).
We then performed cell wound healing assay and found that the addition of ADSC-EXO significantly promoted HSF migration after 12 and 24 h compared with the control group, while the combination group with BTX-A showed the best promotion of HSF migration in vitro (Figure 2C-E).
On the 5th day, HSF treated with ADSC-EXO or ADSC-EXO + BTX-A significantly accelerated wound healing compared with the control cells (Figure 3A).On Day 13, the ADSC-EXO + BTXA treated cells showed significant closure of the wound area with an increase in the average healing rate.Our in vivo data also revealed that the ADSC-EXO + BTXA treatment significantly enhanced re-epithelization compared with the control rats, showing the recovery of hair follicles and sebaceous glands (Figure 4A).In addition, the wound epidermis was thickened, and dermal collagen was evenly distributed and less cross-linked in EXO + BTXA group (Figure 4B).After EXO was applied, the number of new capillaries was significantly increased (Figure 4C).The EXO group and EXO + BTXA group exhibited marked few of dermal collagen density compared with the PBS control.So skin treated with EXO + BTXA inhibits scar formation (Figure 4D).Furthermore, the collagen fibers were much denser and somewhat disorganized in the PBS control.

| ADSC-EXO plus with BTX-A manipulation of VEGFA and type I and III collagen expression in vivo
Afterward, we analyzed the changed level of VEGFA in HSF.We found that VEGFA expression was significantly upregulated after ADSC-EXO, BTX-A, and/or their combination, with the highest combining effect on late-stage wound repair (Figure 5A,B).On Day 16 after surgery, immunohistochemical and real-time PCR data showed that ADSC-EXO plus BTX-A dramatically induced level of VEGFA mRNA expression (Figure 5C-E).Furthermore, we also noticed that compared with PBS control, expression of COL III and COL I in Group ADSC-EXO plus BTX-A was significantly reduced (Figure 6A-C) with an increase in the COL III/COL I ratio (Figure 6D), suggesting that ADSC-EXO and BTX-A treatment decreased collagen deposition, alleviated scar formation, and increased the ratio of COLIII and COLI in vivo.

| ADSC-EXO plus BTX-A regulation of TGFβ1 and TGFβ3 expression in late staged wound repair in vivo
We then analyzed TGF-β3 expression in the skin tissues and found that Group ADSC-EXO and Group ADSC-EXO plus BTX-A upregulated TGF-β3 expression on Day 16 after surgery with more obvious TGF-β3 expression in the combination treatment group than that of Group BTX-A only (p < 0.01; Figure 6).However, TGF-β1 expression in Group BTX-A was downregulated compared with that in Groups ADSC-EXO and ADSC-EXO plus BTX-A (p < 0.01), leading to a higher ratio of the TGF-β3/TGF-β1 in Group ADSC-EXO, Group BTX-A, and Group ADSC-EXO plus BTX-A than in Group PBS (Figure 7A-C).These data suggest that BTX-A could inhibit scar formation by an increase in the ratio of TGF-β3/TGF-β1 (Figure 7D).

| DISCUSS ION
In this study, we performed in vitro and in vivo experiments to demonstrate the synergistic therapeutic effects of the combined ADSC-EXO and BTX-A on skin wound repair.We first isolated the ADSC-EXO from primarily cultured ADSCs and found that ADSC-EXO possessed a circular vesicle shape with a 30-180 nm diameter.ADSC-EXO and/or BTX-A treatment of HSF significantly accelerated HSF migration in vitro and wound repair capacity in a rat model of skin wound healing.ADSC-EXO and BTX-A treatment also dramatically induced VEGFA expression but reduced COL III and COL I protein expression in vivo.ADSC-EXO and BTX-A treatment significantly upregulated TGF-β3 but downregulated TGF-β1 expression on Day 16 after surgery.In conclusion, our current study demonstrated that ADSC-EXO plus BTX-A treatment synergistically affected skin wound repair by upregulation of VEGF expression and the TGF-β3/TGF-β1 and COL III/COL I ratio.Future study will assess their effects clinically.
Adipose tissue contains several mesenchymal stem cells and could serve as an active endocrine organ to repair soft tissue trauma. 19,20ADSC can regulate cell functions through the host immune system and paracrine signaling pathway rather than direct induction of cell differentiation.ADSC-EXO possesses an important paracrine activity in stem cells. 5Previous studies revealed that ADSC-EXO was able to enhance skin wound healing and skin regeneration, 11 although the underlying molecular mechanism remains to be defined.Furthermore, a previous report assessed the effect of BTX-A injection on the regulation of scar formation and showed a significant effect of BTX-A on wound healing and reduction of scar formation. 21Indeed, our current study further confirmed the effectiveness of both agents on skin wound repair.
There are different clinical treatment options for the repair of a skin wound, including skin and flap transplantation, biological scaffolds, growth factor therapy, and/or laser therapy.However, no standard treatment protocols are available yet. 22Their effectiveness varies clinically and even has certain limitations and side effects.4][25] It is well documented that chronic and dysregulated inflammatory responses might alleviate wound healing, enhance tissue fibrosis and scar formation, or inhibit re-epithelialization during the inflammatory phase of the acute healing process. 26During wound repair, macrophages that can secrete TGFβ are the most crucial inflammation cells in the site, which play a pivotal role in skin regeneration, while macrophages transform into M1-type or M2-type macrophages in the early and late stages of wound repair, respectively. 27M1 macrophages promote tissue inflammation, whereas M2 macrophages decrease inflammation and encourage tissue repair. 28Cell proliferation, tissue neovascularization, collagen deposition in the wound site, granulation tissue formation, re-epithelialization, and wound contraction may occur in order during the wound repair process. 29However, macrophage dysfunction could result in excessive inflammation and/ or tissue fibrosis.The formation of tissue neovascularization is also pivotal in wound healing and tissue repair. 30,31In the final stage of tissue remodeling, fibroblasts will transform into myofibroblasts to accelerate skin re-epithelialization to close the wound surface.After that, both fibroblasts and myofibroblasts undergo apoptosis, but they may excessively secrete irregularly arranged collagen bundles, leading to scar formation. 32In this regard, effective wound treatment with proper agents could successfully lead the repair process.Indeed, ADSC has been confirmed to promote wound healing through its paracrine function, while EXO, as an essential paracrine factor, plays a crucial role in wound repair.Many studies have reported that fibroblasts can internalize ADSC-EXO to stimulate cell proliferation, migration, and collagen synthesis in a dose-dependent manner, 24 while fibroblasts participate in soft tissue wound healing 5 through their migration, proliferation, and collagen synthesis capacities. 33Our current study showed that ADSC-EXOs could enter into HSF cytoplasm, and adding ADSC-EXO and BTX-A into HSF culture enhanced HSF migration, the first action in wound repair.5][36] Indeed, our current data showed that ADSC-EXO significantly induced VEGFA expression and TGFβ level.As we know, TGFβ1 can stimulate fibroblast secretion of the cell matrix to accelerate wound healing into the proliferation stage, evidently by expression of TGFβ1 at first and then falling back during the wound repair process. 37Our current data showed ADSC-EXO did not significantly upregulate TGFβ1 expression after wound healing entered the proliferation phase on Day 16.Moreover, TGFβ3 has been associated with anti-fibrosis or scar-free wound healing, which was reported to play an essential role in regulating the movement of the epidermis and dermal cells during wound repair. 38TGF-β3 has been used to prevent and reduce scarring [39][40][41] as a negative regulator of myofibroblastic phenotypes.
In this regard, EXO may promote wound healing by a decrease in healing time in the early stage, while EXO may inhibit the synthesis of collagen by reducing scar formation in the later stage. 42EXO can regulate the type I and III collagen ratio and TGF-β3/TGF-β1 and optimize fibroblast properties, like promoting fibroblast migration and proliferation to accelerate soft tissue wound healing. 38We found that ADSC-EXO induced TGF-β3 expression and optimized collagen deposition, shortened wound healing time, and reduced scar formation, downregulating the expression of type I and type III collagen but upregulating the ratio of type III and type I collagen.BTX-A, a neurotoxic protein, can inhibit acetylcholine release at the neuromuscular junction, resulting in muscle paralysis that lasts up to 6 months. 43The synaptosome-associated protein (25 kDa in molecular weight) can be cleaved by heavy chain binding to the nerve endings, triggering internalization by the endocytosis and inhibition of the acetylcholine release; thus, BTX-A was widely used in the clinic for various human diseases, including blepharospasm and facial dysfunction. 44While BTX-A can reduce scar formation, 45,46 Ziade et al. 47 demonstrated that BTX-A effectively reduced inflammatory cell infiltration and fibroblasts and TGF-β1 expression in wounds in vivo, further supported by our current data.
In conclusion, treating skin wounds with ADSC-EXO and BTX-A was an effective and innovative anti-scarring therapy for skin wound healing.ADSC-EXO plus BTX-A accelerated wound repair at the early stage and reduced scar formation later by increasing vascular nutrition, reducing proliferation, increasing apoptosis, and inhibiting excessive collagen deposition.However, our current study has some limitations.For example, the in vivo model may antibodies for 2 h.Next, the membranes were washed with TBS-T, and the positive protein signals were detected using the enhanced chemiluminescence (ECL) reagents (Coolaber).The antibodies used were an anti-VEGFA (Cat.# ab214424; Abcam), anti-TGF β1 (Cat.# ab215715; Abcam), anti-TGF β3 (Cat.# AF-243-NA; Bio-techne), anti-COL I (Cat.# GB114197; Servicebio), COL III (Cat.# GB11023; Servicebio), anti-rabbit IgG (Cat.# GB23303; Servicebio), and antigoat IgG (Cat.# GB23404; Servicebio) at a dilution suggested by the manufacturers.Positive protein signals were captured using Amersham Imager 600 (GE Healthcare) and quantified using the Image Quant TL software against a loading control (GADPH protein).

F I G U R E 1
Characterization of ADSC and EXO.(A) ADSC morphology under microscope.Scale bar = 200 μm.(B) Immunofluorescence analysis of CD29 in ADSC.(C, D) Flow cytometry.It detects the surface molecule in ADSC.Negative CD45 and CD31 were detected for an average expression rate of 30.6%, while positive CD29 was detected for an average expression rate of 87.7%.(E) Stained with alizarin red.(F) Stained with Alcian blue.(G) Stained with oil red O. (H) The transmitted electron microscopy.The images of ADSC-EXO and EXO showed round vesicles.Scale bar = 200 μM.(I) The distribution of ADSC-EXO size with the largest size of 120 nm diameter.(J, K) The NTA analysis.The data show the concentration and size of EXO after The NTA analyzer.(L) The exosomes' marker proteins (Alix) detected by Western blot.

was 1 . 1 ×
10 10 /mL analyzed using the ZetaView nanoparticle tracker software (Figure 1J,K), positive expression of exosome-specific proteins, indicating that our isolated ADSC-EXO index was consistent with the essential characteristics of the exosomes.F I G U R E 2 Effect of EXO and BTX-A on HSF migration ability.(A) HSF morphology under a light microscope.Scale bar = 200 μm.(B) Immunofluorescence.Under the microscope, green fluorescence could be accumulated in fibroblast cytoplasm after adding PHK67-labeled EXO into HSF culture for 2 h.Scale bar = 100 μm.(C) Wound healing assay.After ADSC-EXO, BTX-A, and their combination treatment for 0, 12, and 24 h, HSF were subjected to the wound healing assay.Scale bar = 500 μm.(D and E) Statistical data of the wound healing assay at 12 h (D) and 24 h (E).**p < 0.01 and ***p < 0.001 versus the control; ∆ p < 0.05 and ∆∆ p < 0.01 between BTX-A and ADSC-EXO + BTX-A; # p < 0.05 and ## p < 0.01 between ADSC-EXO and ADSC-EXO + BTX-A.
Botulinum toxin A on skin wound healing in vivo.(A) Wound healing after injection of ADSC-EXO, BTX-A, or their combination in skin wound repair.(B) Percentage of the wound area after treatment of skin trauma with ADSC-EXO and BTX-A.**p< 0.01 and ***p < 0.001 compared to control; # p < 0.05 and ## p < 0.01 between ADSC-EXO and BTX-A; and ∆ p < 0.05 and ∆∆ p < 0.01 between BTX-A and ADSC-EXO + BTX-A.

F I G U R E 4
EXO promotion of capillary formation and collagen production at the wound sites.(A) H&E staining of skin tissue specimens dissected from the wounding site after 16-day ADSC-EXO and BTX-A treatment.(B) EXO increased in capillary formation at wound site.(C) Capillary quantity statistics.(D) Masson's trichrome staining.(E) Quantification of dermal collagen density.**p < 0.01 and ***p < 0.001 compared to the control; ∆∆∆ p < 0.001 between BTX-A and ADSC-EXO + BTX-A groups.

F I G U R E 5
Effect of EXO and Botulinum toxin A on VEGFA expression in a rat model of skin wound repair.(A) Western blot.Tissue specimens were obtained from the late stages of treatment.(B) Quantified data of A. (C) Immunohistochemistry.The expression of VEGFA protein was analyzed using immunohistochemistry in skin tissues of ADSC-EXO and BTX-A treatment in late skin wound repair.(D) Summarized data of B. (E) Level of VEGFA mRNA was upregulated in the EXO + BTX-A group.**p < 0.01 and ***p < 0.001 compared to the control; # p < 0.05 and ## p < 0.01 between ADSC-EXO and ADSC-EXO + BTX-A groups; ∆∆ p < 0.01 and ∆∆∆ p < 0.001 between BTX-A and ADSC-EXO + BTX-A groups.

F I G U R E 6
Effect of ADSC-EXO and Botulinum toxin A on collagen levels in late skin wound tissues.(A) Western blot.Levels of COL I and COL III proteins were assayed using Western blot in skin tissue samples.(B and C) Quantified data of A. (D) Calculation of the COLIII/COLI ratio.not represent human skin wound repair.Moreover, the effects and potential mechanisms of ADSC-EXO and BTX-A on wound healing progression are still in their infancy.Further studies are needed to better understand how combining the two regulates wound healing progression.In addition, adding film or some other relevant material could be better to show and evaluate the wound closure/healing, eliminating shrinkage and drying of the wound.Thus, a prospective study is needed to clinically verify these two agents' effectiveness.Additional studies are also needed to investigate the specific mechanisms of action of MSC-EXO and BTX-A in wound healing.E TH I C S S TATEM ENT Animal sacrifice and collection were performed according to the guidelines of the China Laboratory Animal Nursing and Use Regulations and with the approval of the Animal Ethics Committee of Hainan Medical University.

F I G U R E 7
Effect of EXO and Botulinum toxin type A on TGFβ3 and TGFβ1 expression in late skin wound tissues (Day 16).(A) Western blot.The level of TGFβ1 and TGFβ3 proteins was analyzed using Western blot in skin tissues.(B and C) Quantified data of A. (D) Calculation of the TGFβ3/TGFβ1 ratio.**p < 0.01 and ***p < 0.001 compared to the control; # p < 0.05 and ## p < 0.01 between ADSC-EXO and ADSC-EXO + BTX-A; ∆ p < 0.05, ∆∆ p < 0.01, and ∆∆∆ p < 0.001 between BTX-A and ADSC-EXO + BTX-A groups.