Human bone marrow mesenchymal stem cells-derived exosomes stimulate cutaneous wound healing mediates through TGF-β/Smad signaling pathway

Background: Cutaneous wound healing represents a morphogenetic response to injury, and is designed to restore anatomic and physiological function. Human bone marrow mesenchymal stem cells-derived exosomes (hBM-MSCs-Ex) is a promising source for cell-free therapy and skin regeneration. Methods: In this study, we investigated the cell regeneration effects and its underlying mechanism of hBM-MSCs-Ex on cutaneous wound healing in rats. In vitro studies , w e evaluated the role of hBM-MSCs-Ex in the two type s of skin cell s : human keratinocytes (HaCaT) and human dermal fibroblasts (HDFs) for the proliferation . For in vivo studies , we used a full-thickness skin wound model to evaluate the effects of hBM-MSCs-Ex on cutaneous wound healing in vivo . Results: The results demonstrated that hBM-MSCs-Ex promote both two type s of skin cell s growth effectively and accelerate the cutaneous wound healing. Interestingly , we found that hBM-MSCs-Ex significantly down-regulated TGF-β1, Smad2, Smad3, and Smad4 expression, while up-regulated TGF-β3 and Smad7 expression in the TGF-β/Smad signaling pathway . Conclusions: Our findings indicated that hBM-MSCs-Ex effectively promote the cutaneous wound healing through inhibiting the TGF-β/Smad signal pathway . The current result s providing a new sight for the therapeutic strategy for the treatment of cutaneous wounds.


Introduction
Cutaneous wound healing is characterized by repairing damaged tissue and the tissue regeneration orchestrated by multiple cells to re-establish a protective barrier [1]. The primary goals of skin wounds treatment considering rapid wound closure and scar-less healing process. Previous studies have shown that mesenchymal stem cells (MSCs) capable of self-renewal and multipotential differentiation capabilities, which shows promising therapeutic potential for tissue regeneration and skin function recovery [2,3]. However, MSCs transplantation may induce immunoreactivity to the host [4]. To avoid host-immunoreactivity, MSC-derived exosomes shows a alternative therapeutic attention for regenerative wound repair [5][6][7]. The current study has been demonstrated that MSCderived exosomes accelerated the wound healing through increased re-epithelialization, 3 angiogenesis, and tissue granulation [6]. In our previous publication, we have demonstrated that hBM-MSCs-Ex treatment significantly reduced liver fibrosis in rats [8]. In this current study, we used the promising exosomes type, i.e. hBM-MSCs-Ex, to investigate the wound healing process, and to investigate the associated signal pathway mechanism.
TGF-β/Smad signal pathway is an evolutionarily conserved pathway with numerous functions ascribed [9]. Transforming growth factor-beta (TGF-β) is considered as one of the essential growth factors in the natural wound healing process by TGF-β/Smad pathway [10]. Smad is well known as the major inducer of fibroblast differentiation, which is an essential factor for wound healing [11]. During this process, TGF-β activates downstream mediators such as Smad2 and Smad3, which result in the linage differentiation of fibroblasts into alpha-smooth muscle expressing (α-SMA) myofibroblasts [12]. The phosphorylated Smad2/Smad3 activates Smad7, which promoter to up-regulate Smad7 expression in the differentiation process, and thereby Smad7 inhibits TGF-β1 expression for negative feedback regulation [13]. Recent studies have demonstrated that the TGF-β/Smad signal pathway plays a essential vital role in cutaneous wound healing via regulating the proliferation and migration of keratinocytes, dermal fibroblasts, and other skin cells in the affected areas to participate in the wound healing process [14,15]. Furthermore, TGF-β/Smad signaling can regulate tissue fibrosis and scar formation [16]. In this current study, we hypothesized that hBM-MSCs-Ex could promote the cutaneous wound healing process via regulating the TGF-β/Smad signaling pathway.

Cell culture and exosome purification
HaCaT and HDFs purchased from the Chinese Academy of Medical Sciences, China. Human bone marrow mesenchymal stem cells (hBM-MSCs) were generously provided by Dr. Yi Wang (Jilin University, Changchun, China), P3-5 lines of hBM-MSCs were used in the experiments. Cells cultured in DMEM (Gibco, Grand Island, USA) supplemented with 10% FBS (Gibco, Grand island, USA), humidified 5% CO 2 atmosphere at 37 °C. The purification of hBM-MSCs-Ex involves several centrifugation steps, as described previously [17]. Briefly, hBM-MSCs were cultured in serum-free medium (SFM, Gibco, Grand island, USA) for 2 days. Conditioned medium was first filtered using a 0.1-μm filtering unit. The supernatant was concentrated with a 100-kDa molecular weight cutoff (MWCO) hollow fiber membrane (Millipore, Billerica, MA, USA), at 1000g for 30 min. Then, the concentrated supernatant was loaded onto a 30% sucrose/D 2 O cushion (5 ml, density 1.210 g/cm 3 ), and ultracentrifuged at 100,000g for 3 h. After exosome-enriched fraction was collected, washed three time with fresh PBS by centrifuged at 1500g (30min each wash) with 100-KDa MWCO. Finally, purified exosomes were passed through a 0.22-μm filter and stored at −80°C until further use. The protein concentration of exosomes was measured by bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China).

Cell proliferation assay
HaCaT and HDFs cells were cultured until 70~80% confluence, trypsinized cells were plated in 96-well plates at a density of 4,000 cells per well. Briefly, hBM-MSCs-Ex purified and characterization as per previously published methods [8]. The cells were treated with either hBM-MSCs-Ex (25 μg/mL) or PBS (control) (Invitrogen, Shanghai, China), then followed by incubated at 37°C with 5% CO 2 for 5 days.
The cell viability was determined by CCK-8 kit (Sigma, San Francisco, U.S.), and corresponding OD value measured at the 490 nm wavelength. Result expressed as

Immunofluorescence staining (IF)
HaCaT and HDFs cultured by hBM-MSCs-Ex (25 μg/mL) or PBS were incubated in 24-well plate coated coverslip for 24 h. When cells reached 60~70% confluence, plate were washed with PBS and incubated with 4% paraformaldehyde for 10 minutes (RT). The processed cells blocked with 1% counted in ten random optical fields by using ImageJ software.

Animals and treatments
The 8-week old female Sprague-Dawley (200 g) rats were purchased from Jilin Biotechnology Co., Ltd.
(Changchun, China). All animal experiments were performed in accordance with the guidelines of the Animal Experiment Ethics Committee of Jilin University. The animal model was generated according to previously published methods [18]. Briefly, rats were anesthesia and shaved the dorsal hair, followed by full-thickness skin excisional wound were made of about the size of 10mm in diameter circular holes in rat. The rats were randomly divided into three groups (n 8/group): PBS group, hBM-MSCs group (intravenous injection with 1×10 6 cells/ rat); hBM-MSCs-Ex group (250μg, multi-directional subcutaneous injection). The recovery of skin damage were recorded photographically every four days for 16 days. The wound area was measured using lasso tool (Adobe Photoshop CS6), The wounded area were trace and circle the edge of wound on photograph, then calculate the circled area based on the pixels of that area. End of the study, the rats were euthanized on 16 th day to collect the healed and unhealed tissue area in the different treatment groups.

Histological examination
Skin tissue were collected from the mechanical injury region on 16 days, and samples were fixed in Next day, these sections were incubated with biotinylated goat-anti-rabbit IgG antibody for 2 h and incubated with avidin peroxidase reagent sequentially. Then, the sections were incubated with 6 diaminobenzidine solution as the chromogenic agent at 37 °C for 5 min. Finally, we used H&E staining for counterstaining the sections. These sections were photographed using a bright-field microscope (EVOS, Thermo Scientific, Waltham, U.S.). Cutaneous appendage separated by tissue on one microscope sphere was counted as one unit. The α-SMA and VEGF positive area calculation is based on the ratio of positive area /total area of the observed field. We used 6 random fields per section and 6 sections in total (n=8 rats) for the quantification of IHC images.

Western blot
Proteins were extracted from the skin healed tissue in the in SDS sample lysis buffer. The samples were heated to 95°C for 10 min, and 40 μg protein samples were separated on SDS-polyacrylamide gels (5% stacking gel and 12% separation gel). Resolved proteins were then transfered onto nitrocellulose membranes and blocked in 5% nonfat powdered milk for 1h, and probed with respective antibody against TGF-β1, TGF-β3, Smad2, Smad3, Smad4, Smad7 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control (1:1000 dilution, Abcam, Cambridge, UK) overnight at 4°C. The blots were blocked in secondary HRP-conjugated goat anti-rabbit IgG antibodies (1:1000 dilution, Abcam, Cambridge, UK), and visualized by chemiluminescent detection substrates (Immobilon western chemiluminescent HRP substrate, Millipore). The densitometric quantification were performed on the protein bands by using AlphaEaseFC software (Alpha Innotech).

Real-time PCR assay
Total RNA from skin healed tissue was extracted with Trizol (Invitrogen, Shanghai, China) according to the manufacturer's protocol. Briefly, the first-strand cDNA was synthesized with 1μg of total RNA using SuperScript II (Invitrogen). SYBR Green I dye was used for reverse transcription in an ABI 7500 fluorescence quantitative PCR instrument, and the mRNA levels of TGF-β1, TGF-β3, Smad2, Smad3, Smad4, Smad7, and GAPDH were measured using the respective primers listed in Table   (Supplementary Table S)1. The thermocycler conditions as follow: initial step at 95°C for 2min, followed by 40 cycles at 95°C for 15s, and 60°C for 1min. Expression levels were recorded as cycle threshold (Ct). Data were acquired using the 7500 Software (Applied Biosystems Life Technologies, Foster City, CA, U.S.). All reactions were performed in triplicate, and the data were analyzed using the 7 2 -ΔΔCt method.

Statistical analysis
Statistical analysis was performed using Prism 6 (Graph Pad software) and Image J. One-way ANOVA with post-hoc Dunnett's multiple comparisons test were used to test for statistically significant differences between the groups. All quantitative data were given as the mean ± SD and data were acquired by at least three independent experiments, and p<0.05 was considered to be statistically significant.

In vitro, we investigated whether hBM-MSCs-Ex treatment can stimulate the cell proliferation by using
HaCaT and HDFs skin cells. After the procedure, the proliferation assay was measured consecutive for 5-day. In HaCaT and HDFs cells, we observed significant increase in exponential cell proliferation and the growth rate constantly increased from day 2 after the hBM-MSCs-Ex treatment, compared to the PBS group ( Fig.1a and 1b, p<0.05 and p<0.01). Based on the CCK-8 results, we performed immunofluorescent staining analysis on day 5 to confirm whether hBM-MSCs-Ex treatment can induce the proliferative effect in these cell line. As shown in the representative images in Fig.1c

Invivo studies: hBM-MSCs-Ex improves cutaneous wound healing
To investigate the roles of hBM-MSCs-Ex in wound healing, we established a full-thickness skin wounds injury model in rats, as illustrated in the experimental design in Figure 2a. The area measurements in the wounded region show a significant improvement in the wound closure after the treatment of hBM-MSCs-Ex and hBM-MSCs treatment group (Fig 2b and 2c). We observed a significant  (Fig 3a and 3c).
Consist with the above results, the percentage of VEGF positive area was significantly increased in hBM-MSCs-Ex group (12.3% ± 2.4), compared to the PBS group (1.6% ± 1.5/ HPF, p<0.001) and hBM-MSCs group (5.1%± 1.7, p<0.001), (Fig 3a and 3d). The above results indicated that hBM-MSCs-Ex not only enhanced the wound process but also restore skin function and angiogenesis in the affected areas.

hBM-MSCs-Ex regulate the TGF-β/Smad signal pathway
Finally, we investigates the underlying mechanism of hBM-MSCs-Ex induced skin healing process in the affected tissue. The expression level of TGF-β1, TGF-β3, Smad2, Smad3, Smad4, and Smad7 (components of the TGF-β/Smad signaling pathway) were analyzed by Western blot and RT-qPCR.
Both Western blot and RT-qPCR results showed the significantly decreased (respective protein and RNA levels) expression of TGF-β1, Smad2, Smad3, and Smad4 in hBM-MSCs-Ex treatment group, compared the other two control groups (p <0.05, p <0.01). The alteration of signaling molecule during the healing the process illustrated that hBM-MSCs-Ex treatment plays a major mechanism in inhibiting TGF-β/Smad signaling pathway ( Fig.4a and 4b).
To understand the mechanism how the TGF-β/Smad signaling was inhibited. We study the role of Smad7 (downstream molecules) in the TGF-β signaling pathway, interestingly, we detected the Smad7 was significantly increased in hBM-MSCs-Ex treatment group, compared the other two control groups ( Fig.4a and 4b, p <0.05, p <0.01). Inhibitory Smad7, which are activated by the binding of the TGF-β super family to the cell surface receptors. TGF-β1 is fibrotic isoform, while TGF-β3 is the antifibrotic isoform. The hBM-MSCs-Ex treatment group significantly up-regulated the expression of TGF-β3, compared the other two control groups ( Fig.4a and 4b, p<0.05, p<0.01 Recently, studies have shown that the prospective application of MSCs-derived exosomes promoted cutaneous wound healing [5,7,19]. The possible roles of MSCs-derived exosomes in wound healing through promotion of cell proliferation, migration, differentiation, angiogenesis and matrix reconstruction [6]. HaCaT and HDFs are the two major skin cells types which participating in cutaneous wound healing [20]. In our study, we found that hBM-MSCs-Ex promote HaCaT and HDFs growth effectively (Fig1). It indicated that hBM-MSCs-Ex promote HaCaT and HDFs cell proliferation to participate in the process of wound healing. In addition, fibroblasts are critical players for exosomes in wound healing and are the main cell types that synthesize, secrete, and deposit ECM collagen and elastic fibers [21]. Studies have shown that MSCs exert their therapeutic effect via secretion of soluble factors, included the exosomes [5,6]. There is substantial evidence that exosomes have the ability to promoted skin regeneration when applied topically or injected systemically [22,23]. In this study, we have administration subcutaneous injection of exosomes at multi-directional to delay its clearance in the body. In addition, hBM-MSCs appear to be limited because of poor cell retention at the wound site [24], To overcomes this issue we adopted intravenous injection routes to maximize the effect of treating wounds in our treatment groups.  [6,19]. Interestingly, we also found there are many cutaneous appendages regeneration, such as hair follicles and sebaceous glands (Fig3a and 3b). During the normal healing process in addition if provides hBM-MSCs-Ex which favor conditions for the restoration of skin function much quicker than normal healing process.

MSCs
Recently studies, indicated that different types of MSCs therapy or combined therapies with some unique biotechnology factors can stimulate cutaneous wound healing [28][29][30][31]. Current studies have report that a new combination therapeutical approach shows effective combinational treatment composed of adipose derived mesenchymal stem cells (AD-MSCs) platelet-rich plasma (PRP) and hyaluronic acid (HA) dressing which shown to stimulate the wounds healing and regeneration process [30][31][32]. The complete closure of wound is inducing a new neodermis and stimulating regeneration and a protected environment in a humid environment [33][34][35]. Many supporting evidence indicates that adipose-derived stem cells (ASCs) and adipocyte-secreted exosomal microRNA promote wound repair [36]. Another study has demonstrated that hBMSCs on gelatin scaffold with poly Nisopropylacrylamide (pNIPAAm) as transplanted grafts for improving skin regeneration [42]. More recent clinical trials shown to improve the hair density by administration human follicle stem cells (HFSCs) [37][38][39]. In addition, autologous fat grafting it is better approach to consider for the correction of wound scars in the affected regions [40,41]. However, in our current study, we administration directly hBM-MSCs-Ex on mechanical damaged skin area, which is more clinically transferable, more safe and effective cell-free reagents compared with their parents cells skin regeneration therapies.
Moreover, we have demonstrated that, hBM-MSCs-Ex treatment is more effective than of hBM-MSCs in wound healing, such as some indicators of wound area and cutaneous appendages.
TGF-β1/Smad pathway is an important pathogenic mechanism in wound healing [9]. TGF-β1 is considered to be a key mediator in tissues scarring and mostly by activating its downstream against decapentaplegic (Smad) signaling [13]. It has proven that TGF-β1 exerts its biological effects by activating downstream mediators, including Smad2 and Smad3 [15]. The phosphorylates cytoplasmic mediators, Smad2 and/or Smad3, and a heterotrimeric complex is formed with Smad4 that translocate into the nucleus, binds a consensus sequence, and regulates gene transcription [14].
While these activities is negatively regulated by Smad7 expression [16]. In our study, we found hBM-MSCs-Ex significantly down-regulate of TGF-β1, Smad2, Smad3, and Smad4 expression, and up-

Consent for publication
Not applicable.

Availability of supporting data
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Competing interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
This work was supported by the National Natural Science Foundation of China (No. 81570360).

Authors' Contributions
T.J. carried out the molecular genetic, animal studies, conceived of the study, and participated in its design and coordination and helped to draft the manuscript. Z.W. carried out the WB and animal studies. J.S. participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.

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