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% CO2 atmosphere at 37 °C. The purification of hBM-MSCs-Ex involves several centrifugation steps, as described previously . 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/D2O cushion (5 ml, density 1.210 g/cm3), and ultra-centrifuged 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 . 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% CO2 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% bovine serum albumin (BSA; Biosharp, Hefei, China) for 30 minutes. The cells were incubated with primary antibodies against Rb anti-PCNA (1:100 dilution, BD Biosciences, Franklin Lakes, NJ, U.S.), and isotype-matched rabbit IgG/IgM (1:100 dilution, Abcam, Cambridge, UK) served as the negative controls. Anti-rb-FITC-488 secondary antibody (1:500 dilution, Abcam, Cambridge, UK) for 2h, and the nuclei were labeled with DAPI (Thermo Scientific, Waltham, U.S.) for 5min. Images have acquired by a fluorescence microscopy (EVOS, Thermo Scientific, Waltham, U.S.), and the PCNA positive cells were 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 . 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 1×106 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 16th day to collect the healed and unhealed tissue area in the different treatment groups.
Skin tissue were collected from the mechanical injury region on 16 days, and samples were fixed in 10% formalin in PBS and embedded in paraffin. The skin tissue were sectioned at 4-μm thickness to perform Hematoxylin and eosin (H&E) staining. The staining were performed following the manufacturer’s protocols (Sigma, San Francisco, U.S.). The sections were processed for immunohistochemistry (IHC) using the Kit (Maixin KIT-9710, Fuzhou, China) following the manufacturer’s instructions. Briefly, the sections were deparaffinized, antigen retrieval were performed by immersion slides in 0.01M sodium citrate buffer solution for 15 min. Quenching of endogenous peroxidase by processing the sections in 3% H2O2 for 15 min, followed by blocked sections with 10% normal goat serum for 1 h at 37 °C. The sections were incubated with primary antibody anti-α-SMA or anti-VEGF with 1:500 dilution (Abcam, Cambridge, UK) for overnight at 4 °C. 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 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.
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 2-ΔΔCt method.
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.