Human bone marrow mesenchymal stem cells-derived exosomes stimulates cutaneous wound healing thourgh TGF-β/Smad signaling

Background: Cutaneous wound healing represents a morphogenetic response to injury, and it 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 therapeutic effects and underlying mechanism of hBM-MSCs-Ex on cutaneous wound healing in rats. We assessment of the role of hBM-MSCs-Ex in the two type of skin cell: human keratinocytes (HaCaT) and human dermal fibroblasts (HDFs). proliferation in vitro . Furthermore, we used a full-thickness skin wounds to evaluate the effects of hBM-MSCs-Ex on cutaneous wound healing in vivo. Results: Our results demonstrated that hBM-MSCs-Ex promote both two type of skin cell growth effectively and accelerate the cutaneous wound healing ( p <0.01). Then, we found that hBM-MSCs-Ex significantly down-regulated TGF-β1, Smad2, Smad3, and Smad4 expression, while up-regulated TGF-β3 and Smad7 expression ( p <0.05). Conclusions: In conclusion, our findings indicated that hBM-MSCs-Ex effectively promote the cutaneous wound healing through inhibiting the TGF-β/Smad signal pathway, providing a new sight for the therapeutic strategy of hBM-MSCs-Ex for the treatment of cutaneous wounds.

cell viability was examined by CCK-8 (Sigma, San Francisco, U.S.), and corresponding OD value measured at the 490 nm wavelength.

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 Ethic Committee of Jilin University. The animal model was made according to previously published methods [17]. Briefly, rats were anesthesia and shaved the dorsal hair. Then, we made one full-thickness skin excisional wounds of 10 mm in diameter circular holes in each rats. 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 (250 mg, harvested from 1 × 10 6 hBM-MSCs).
The skin damage was treatment and recorded photographically every four days. The wound area was calculated out using Adobe Photoshop CS6, lasso tool was used to trace and circle the edge of wound on photograph, then calculate the circled area based on the pixels of that area. Rats were euthanized on day 16 and collected the healed tissue.

Histological Examination
Skin tissue sections were cut at 4 µm thickness and used for histological examination. Hematoxylin and eosin (H&E) staining was performed following the manufacturer's standardized protocols (Sigma, San Francisco, U.S.). Immunohistochemistry (IHC) was carried using the Kit (Maixin KIT-9710, Fuzhou, China) following the manufacturer's instructions. Briefly, the sections were deparaffinized, rehydrated and incubated in a 99 °C water bath for 15 min, 3% H 2 O 2 was added for 15 min, and blocked with 10% normal goat serum for 1 h at 37 °C. Next, the sections were incubated with primary antibody anti-α-SMA with1:500 dilution (Abcam, Cambridge, UK) overnight at 4 °C. Next, these sections were incubated with biotinylated goat-anti-rabbit IgG antibody for 2 h. Then, we used diaminobenzidine solution as the chromogenic agent at 37 °C for 15 min, and incubated with avidin peroxidase reagent sequentially. Lastly, we used hematoxylin was for counterstaining. These sections were photographed using a microscope (EVOS, Thermo Scientific, Waltham, U.S.).

Western Blot
Proteins were extracted from the skin healed tissue in thelysis buffer. The protein samples in SDS sample buffer were heated to 95 °C for 10 min, and separated on SDS-polyacrylamide gels. Resolved proteins were then electro blotted onto nitrocellulose membranes and probed with antibody against TGF-β1, TGF-β3, Smad2, Smad3, Smad4, Smad7 and glyceraldehyde-3-phosphate dehydrogenase

Real-time PCR Assay
Total RNA from skin healed tissue was extracted with Trizol (Invitrogen, Shanghai, China) according to the manufacturer's protocol. 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, and the primers were added in supplementary Table S1.
The thermocycler conditions as follow: initial step at 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. 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 2 −ΔΔCt method.

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

Assessment of the role of hBM-MSCs-Ex in the two type of skin cell proliferation
In vitro, we investigated whether hBM-MSCs-Ex stimulate two type of skin cell proliferation, both HaCaT and HDFs proliferation was measured for 5-day. As shown in the Fig. 1a and 1b, the growth rate of hBM-MSCs-Ex treatment group significantly increased both in HaCaT and HDFs, compared of PBS group (p < 0.05 and p < 0.01). Based on the CCK-8 results, we conducted immunofluorescent staining analysis to confirm the proliferative role of the above results. As shown in Fig. 1c and 1d, the intensity of PCNA + in hBM-MSCs-Ex treatment group was significantly higher than that of PBS group (28.2% vs 62.1% in HaCaT and 34.8% vs 71.3% in HDFs) both in the two type of skin cells (p < 0.01).
In vitro experiment, the results demonstrated that hBM-MSCs-Ex promote both two type of skin cell (HaCaT and HDFs) growth effectively.

hBM-MSCs-Ex Promote Cutaneous Wound Healing In Vivo
We established a full-thickness skin wounds injury mode in rats to investigate the roles of hBM-MSCs-Ex in wound healing (Fig. 2a). As shown in Fig. 2b and 2c, the wound area of hBM-MSCs-Ex treatment group was significantly reduced from 4 to16 days after surgery, compared with PBS and hBM-MSCs group (p < 0.05, p < 0.01). It indicating that hBM-MSCs-Ex treatment is not only much better than PBS group (negative control), but also better than hBM-MSCs group (positive control). The wound in hBM-MSCs-Ex group healed completely on the 16 ± 2.3 day after surgery. Taken together, the results showed that hBM-MSCs-Ex effectively accelerated the cutaneous wound healing.
Next, we assessed the effects of the hBM-MSCs-Ex on the wound healing quality. H&E staining results indicated that cutaneous appendages including hair follicles and sebaceous glands in hBM-MSCs-Ex group (21.3 ± 5.4 /filed) were much more than in PBS group (1.2 ± 2.8 /filed, p < 0.001) and hBM-MSCs group (15.4 ± 4.1 /filed, p < 0.001), as shown in Fig. 3a and 3b. α-SMA is an important indicator of angiogenesis. IHC results showed that the percentage of α -SMA positive area was significantly increased in hBM-MSCs-Ex group (8.1% ± 1.2), when compared with the PBS group (1.3% ± 0.5 / HPF, p < 0.001) as well as hBM-MSCs group 5.6%± 0.9, p < 0.01). It indicated that hBM-MSCs-Ex enhanced skin function and angiogenesis.

hBM-MSCs-Ex Regulate The TGF-β/Smad Signal Pathway
To investigate the underlying mechanism of the effect of hBM-MSCs-Ex and on skin healed tissue, the expression level of TGF-β1, TGF-β3, Smad2, Smad3, Smad4, and Smad7 (components of the TGFβ/Smad signaling pathway) were analyzed by Western blot and RT-qPCR. Both Western blot and RT-qPCR results showed the significantly decreased 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), which illustrated the inhibition of TGF-β/Smad signaling in the presence of hBM-MSCs-Ex ( Fig. 4a and 4b). To find how the TGF-β/Smad signaling was inhibited, 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 anti-fibrotic isoform. Interestingly, 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). These results suggest that hBM-MSCs-Ex effectively promote the cutaneous wound healing through inhibiting the TGF-β/Smad signal pathway . In our study, we found that hBM-MSCs-Ex promote HaCaT and HDFs growth effectively (Fig. 1). It indicated that hBM-MSCs-Ex may promote HaCaT and HDFs proliferation to participate the process of the wound healing. In addition, fibroblasts are key players for exosomes in wound healing, and are the main cell types that synthesize, secrete, and deposit ECM collagen and elastic fibers [20].
MSCs-derived exosomes accelerate wound healing through promoting angiogenesis and restoration of skin function [21]. Previous study has proved that stem cell conditioned medium may contains many pro-angiogenesis factors to promoted wound healing in skin injury [22,23]. Our results demonstrate that hBM-MSCs-Ex significantly accelerate wound healing (Fig. 2b and 2c), and promoted α-SMA expression which is an important indicator of angiogenesis ( Fig. 3a and 3c). It due to that MSCsderived exosome contains various growth factors to play an important role in cutaneous regeneration and repair [6,18]. Interestingly, we also found there are many cutaneous appendages regeneration, such as hair follicles and sebaceous glands ( Fig. 3a and 3b). This provides favorable conditions for the restoration of skin function.
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 mothers to against decapentaplegic (Smad) signaling [13]. It have prove 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 translocates into the nucleus, binds a consensus sequence, and regulates gene transcription [14].
While these activity 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 upregulate of Smad7 expression (Fig. 4). It demonstrated that hBM-MSCs-Ex may accelerate cutaneous wound healing through inhibiting the TGF-β/Smad signal pathway (Fig. 5). TGF-β1 are associated with fibrosis, while TGF-β3 has been associated with anti-fibrotic or scar-less in wound healing activity, and they have been observed to play an ssential role in regulating epidermal and dermal cell movement during wound repair[10]. In our study, we found hBM-MSCs-Ex decreased of TGF-β1 expression and increased TGF-β3 expression (Fig. 4). This may be one of the crucial reasons to promotes the skin scar-less wound healing.
In conclusion, we successfully investigated the role of hBM-MSCs-Ex on cutaneous wound healing. Our results demonstrated that hBM-MSCs-Ex can exert promoting effect of cutaneous wound healing via inhibiting the TGF-β/Smad signal pathway.

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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