2.1 Animals:
Fifteen 6-week-old, male, BALB/c mice were purchased from the Guangdong Medical Laboratory Animal Center, All experimental and animal care procedures were approved by the Animal Care and Use Committee of Shenzhen people’s hospital. Animals were randomly divided into three groups to serve three I-R circles (Fig. 2A). Three time points, 1, 7 and 14 days, were evaluated for a total of 15 mice for the entire study. Before all procedures, the mice were allowed to acclimatize to their environment for 1 week.
2.2 Mice skin wound model and treatment
The mice were anesthetized with isoflurane inhalation (1.8%). To create the pressure ulcers wound, the back dorsal of the mice was shaved and cleaned with 75% alcohol subsequently pinched between two circular magnets (Dongguan JinKun Magnet products, CO) that were 10 mm in diameter, 5 mm thick, and strength of 4000 G. The full-thickness skin was pulled up and placed between a pair of the magnet disks. The dorsal skin of mice was exposed to a 12h magnetic pressure and 12h rest (IR cycle). A total of 3 rounds of IR cycles were performed. The compressed area was left uncovered for the rest of the procedure after PU wound induction protocol.
Mice were randomly assigned to 3 different treatment groups and were left untreated (Control) or injected with either 200µL of PBS or 200ug of purified hucMSC exosomes (huCMSC-Exo) re-suspended in 200ul PBS around the wounds at 4 injection sites. All wounds were studied by histopathological analysis at everyday post-wounding. The wound-size reduction was calculated using the equation: wound-size reduction (%) = (AO – At)/AO × 100, where AO is the initial wound area, and At is the wound area at everyday post-wounding. On days 1, 2, and 3 after injection of these DIR-labeled exosomes into the wounds, the fluorescence was measured every 12 hours by bioluminescence imaging.
2.3 Cell Culture
Human umbilical cords mesenchymal stem cells were purchased from Cyagen (stock number: HUXUC-01001 batch number: 160117I31). MSCs were cultured immediately upon receipt using Cyagen's umbilical cord mesenchymal stem cell culture medium (Cyagen, Basal Medium, For Human: Umbilical Card MSCs). Primary culture of MSCs and dermal fibroblasts were established using standard procedures. MSCs and fibroblasts were cultured in an incubator at 37 ° C, 5% CO2. MSCs were sub-cultured at a ratio of 1:3 every 2 or 3 days until passage 5. Medium was then replaced by TM-ACF PLUS Medium Human (MesenCult) supplemented with cell growth supplement (MesenCult TM-ACF PLUS 500X Supplement). MSCs were passaged at a ratio of 1:2 in T175 flasks, and the culture supernatant was collected for exosome purification when the culture confluency reached 90%.
2.4 Exosome purification and identification
At the end of the incubation period, the media were collected and centrifuged at 400× g for 10 minutes,2000× g for 10 minutes, and 10000× g for 30 minutes to remove cell fragments and protein aggregates. Then the supernatant was centrifuged at 100000× g for 120 minutes to pellet the exosomes. In order to purify the exosome, the supernatant was discarded and the exosome pellet was washed with ice-cold PBS follwed by centrifugation at 100000× g for 120 minutes. The washed exosome pellet was re-suspended with PBS and stored at -80 ° C, or directly added to sample loading buffer for purity analysis by Western Blotting. The exosomes were measured for their protein content using the BCA protein assay kit (Pierce Protein Biology; Thermo Fisher Scientific Life Sciences). Exosome purification was confirmed by western blotting detection of exosomal surface markers CD9/CD63/CD81. The shape and size of the exosomes were analyzed by transmission electron microscope (Tecnai G2 Spirit Biotwin with transmission electron microscopy).
2.5 In vitro exosome internalization and effects on fibroblast.
To examine the effect of hucMSC-Exo on fibrosis in vitro, primary fibroblast were treated with purified and DIR labelled hucMSC-Exo (100ul, 1mg/ml) for 12 h. Human fibroblasts and DIR labelled huCMSCs-exo were seeded on coverslips in 96-well plates at 37°C, 5% CO2 in a humidified atmosphere and washed three times subsequently before counterstaining with Hoechst nuclear stain (Hoechst 33342, Germany) for immunofluorescence microscopy imaging. To analyze the effect of DIR labelled hucMSC-Exo on fibroblast activation, fibroblasts were seeded into 24-well plates (5 x 105 cells/well) in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 ng/ml fibroblast growth factor 2, 20% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Fibroblasts were cultured for 3 days either in presence of DIR labelled hucMSC-Exo (0.5mg/ml), PBS, or left untreated (control). Treated fibroblast were collected for mRNA expression analysis by RT-qPCR
2.6 Histology
Cutaneous wound bed tissues were dissected at 14 days and fixed in 4 % phosphate-buffered formalin (pH 7.4), embedded in paraffin, sectioned at 5µm, and mounted on glass slides. The sections were stained with hematoxylin and eosin (H&E) using standard procedures and observed under a light microscope (Olympus BX53)
2.7 Assessment of collagen
Masson’s trichrome staining, which differentially stains collagen blue, was used to evaluate the wound bed for collagen deposition and to assess healing). The mRNA levels of Type I, III collagen and TGF-β were examined by RT-qPCR. Expression levels were normalized to the housekeeping gene GAPDH and translated to relative values. The primers sequence used in this study are : Type I collagen: forward, 5′- TGACCGATGGATTCCCGTTC − 3′, reverse, 5′- GCAGGGCCTTCTTGAGGTTG − 3′, Type III collagen: forward, 5′- AAGCACTGGTGGACAGATTC-3′, reverse, 5′- CGGCTGGAAAGAAGTCTGAG − 3′, GAPDH: forward, 5′- AGCTTGTCATC AACGGGAAG − 3′, reverse, 5′- TTTGATGTTAGTGGGGTCTCG − 3′, TGF-β: forward, 5'-ACAACCCACACCTGATCCTC-3', reverse, 5'-GTTCGTGGACCC ATTTCCAG-3'.
2.8 Immunohistochemistry
To evaluate inflammation and angiogenesis, biotinalyted-antibodies against alpha-smooth muscle actin (α-SAM) (Abcam), endothelial marker CD34 (Abcam) and HMGB1 (R&D Systems) were used to label PU wound sections. To assess the number of vessels, the entire area of the wound on the slide was observed under a microscope and the number of vessels was counted in each of the four quadrants of the wound and the average was calculated. Scoring of staining was done blindly by 4 independent experimenters.
2.9 HMGB1 content measurement
HMGB1 were examined by RT-qPCR using the primer set: forward, 5'-CTTCCTCATCCTCTTCATCC-3', and reverse, 5'-GCCCTATGAGAA GAA AGC TG-3', and GAPDH as an internal standard. For Western blot analysis, the proteins from skin wound tissue were isolated using RIPA lysis buffer. Protein concentrations were measured with the BCA protein assay kit. Anti-HMGB1 (Sigma-Aldrich) antibody was used for western blot analysis. The blots were developed with Western Lightning Plus ECL.
2.10 Statistical Analysis
Group data were expressed as the mean and SD. Data were analyzed using GraphPad Prism version 6 (GraphPad Software). Statistical differences were determined by Student’s t-test or analysis of variance, with p-values < 0.05 considered statistically significant.