ADSCs isolation and cultivation
Human adipose tissue was obtained from patients who had undergone lipoplasty, and all enrolled patients signed the informed consent form. The liposuction sites were bilateral thighs and buttocks. The adipose tissue was rinsed three times with phosphate-buffered saline (PBS, Solarbio, Beijing, China), and then 0.1% type I collagenase (Sigma–Aldrich, St. Louis, MO, USA) was used to digest the adipose tissue for 50 minutes. Centrifuge and filter the mixture after terminating the digestion to obtain ADSCs. ADSCs were cultured in Dulbecco's modified Eagle’s medium:F-12 (DMEM/F-12, Gibco BRL, NY, USA) containing 10% foetal bovine serum (FBS, Gibco BRL, NY, USA) and 1% penicillin/streptomycin (Solarbio, Beijing, China) at 37 °C in 5% carbon dioxide (CO2). The medium was changed every 2 days. Subculture was performed when the cell fusion rate reached 80%-90%. Cells at passage 4 were used in this experiment.
Flow Cytometry to identify cell surface markers
ADSCs were collected and washed three times in PBS and then resuspended in PBS containing 1% bovine serum albumin (BSA, Invitrogen, CA, USA). The cell suspension was incubated with fluorescein isothiocyanate (FITC)-conjugated antibodies against CD90, phycoerythrin (PE)-conjugated antibodies against CD105, CD34, CD31, CD45, and Brilliant Violet421 (BV421)-conjugated antibodies against CD73 at 4 °C for 30 min in the dark, washed twice, resuspended in 2% BSA and detected by flow cytometry (BD Biosciences, CA, USA).
Adipogenic and osteogenic differentiation
ADSCs were inoculated in 6-well plates (Corning, NY, USA) (cell concentration of 2×105/well) until the confluence of cells reached 80%-90%. Adipogenic differentiation was proceeded using basic medium A containing 10% FBS, 1% penicillin-streptomycin, 1% glutamine, 0.2% insulin, 0.1% 3-isobutyl-1-methyl xanthine (IBMX), 0.1% rosiglitazone, and 0.1% dexamethasone for 3 days and basic medium B containing 10% FBS, 1% penicillin-streptomycin, 1% glutamine, and 0.2% insulin for 1 day, and they alternated 4 times (Cyagen Bioscience, Inc., China, HUXMD-90031). Osteogenic differentiation was proceeded using basic medium containing 10% FBS, 1% penicillin-streptomycin, 1% glutamine, 0.2% ascorbate, 1% β-glycerophosphate, and 0.01% dexamethasone for 3 weeks (Cyagen Bioscience, Inc., China, HUXMD-90021).
At the end of induction, 4% paraformaldehyde (Solarbio, Beijing, China) was used to immobilize the cells for 30 min, and Oil Red O and Alizarin Red S dye solutions were used to assess adipogenic and osteogenic differentiation, respectively. The cells were observed under a microscope (Olympus, Tokyo, Japan) after staining.
Chondrogenic differentiation
ADSCs were harvested and resuspended in a centrifuge tube in basic medium containing 0.3% ascorbate, 0.01% dexamethasone, 1% insulin ferro-selenium transporter (ITS) supplement, 0.1% sodium pyruvate, 0.1% proline and 1% transforming growth factor-β3 (TGF-β3) (4×105/tube). The cells were cultured at 37 °C in 5% CO2 for 21 days (Cyagen Bioscience, Inc., China, HUXMD-90041).
After induction, 4% paraformaldehyde was used to immobilize the cartilage balls for 30 min, and Alcian blue staining was used to assess chondrogenic differentiation. The sections were examined under microscope.
HSFs isolation and cultivation
Hypertrophic scar samples were obtained from patients who had undergone plastic surgery (Department of Burn and Plastic Surgery, the Fourth Medical Center, Chinese PLA (People’s Liberation Army) General Hospital. Finally, a total of 8 samples were collected, including 5 males and 3 females with an average age of 35.0±11.7 years. Dermal tissues were washed 3 times with PBS and then minced into pieces (~1 mm). Pieces were explanted in Dulbecco's modified Eagle’s medium (DMEM, Gibco BRL, NY, USA) containing 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO2. Subculture was performed when the cell fusion rate reached 80%-90%. Cells at passage 4 were used in this experiment.
Establish indirect co-cultivation system
HSFs were resuspended in DMEM/F-12 and inoculated into the lower chamber of a Transwell coculture plate (Corning, NY, USA, 3422/3450). ADSCs were resuspended in DMEM/F-12 and inoculated into the upper chamber of the plate. Only the same amount of medium was added to the upper chamber in the blank group. In general, the ratios of ADSCs and HSFs in coculture were 0:1, 0.5:1, 1:1 and 2:1.
Cell proliferation and migration
HSFs were collected after culture with ADSCs for 24 h, 48 h and 72 h. Subsequent steps were performed according to the instructions supplied with the Cell Counting Kit-8 (Beyotime Biotechnology, Shanghai, China), and the absorbance at 450 nm was measured with an enzyme immunoassay analyser (BioRad 680, Hercules, USA).
The migration property was evaluated by scratch assay. HSFs were cultured in a six-well plate, and when 90% confluence was reached, the cells were scratched with a
pipet tip through the well bottom centre. Additionally, ADSCs were added to the upper chamber of the plate as previously described. Images were taken using a microscope (Olympus, Tokyo, Japan) every 24 h. ImageJ software was used to measure the area and length of the scratches to calculate the average width of the scratches. The migration rate of the scratch was calculated as follows: migration rate (%) = (W0 − Wt)/W0 × 100%, where W0 is the original width and Wt is the remaining width at the measured time point.
Cell apoptosis
Cell apoptosis was detected by an Annexin V FITC PI Apoptosis Kit (BD Biosciences, CA, USA). After 48 h of coculture, the HSFs were collected and resuspended in a flow tube with 1× Annexin V binding buffer, and Annexin V FITC and PI were added according to the instructions. After incubation for 15 min, cells were detected by flow cytometry.
ROS evaluation
After 48 h of coculture, HSFs were washed with PBS and incubated with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) in a cell incubator (37 °C in 5% CO2, 30 min) according to the instructions provided by the Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, Shanghai, China). The cells were incubated with Rosup as a positive control, and the probe was omitted as a negative control. ROS were detected by flow cytometry.
Total- superoxide dismutase(T-SOD) activity
A T-SOD assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to detect the T-SOD activity of HSFs after 48 h of coculture based on the autooxidation of hydroxylamine. All procedures were carried out according to the instructions, and the developed colour was measured at 550 nm using an enzyme immunoassay analyser.
Quantitative real-time polymerase chain reaction (qRT–PCR)
Briefly, total RNA was extracted from HSFs after 48 h of coculture with ADSCs using TRIzol (Invitrogen, Carlsbad, CA, USA), and RNA purity was evaluated by calculating the A260/A280 ratio between values of 1.8 and 2.0. The isolated RNA was reverse transcribed into complementary DNA using the Prime Script RT Reagent kit (TaKaRa, Tokyo, Japan). Primers were obtained from Takara Biotechnology. Quantitative PCR was performed using the CFX96TM real-time system (Bio–Rad, Hercules, CA, USA) using SYBR Premix Ex Taq II (Takara, China) in a 12 μl PCR solution. The primer pairs used for gene amplification were as follows: Nrf2: forward GTATGCAACAGGACATTGAGCAAG, reverse TGGAACCATGGTAGTCTCAACCAG, Keap1: forward CATCGGCATCGCCAACTTC, reverse ACCAGTTGGCAGTGGGACAG, NQO1: forward GGATTGGACCGAGCTGGAA, reverse GAAACACCCAGCCGTCAGCTA, GAPDH: forward GCACCGTCAAGGCTGAGAAC, reverse TGGTGAAGACGCCAGTGGA. The results were normalized against the mean Ct values for GAPDH using the ΔCt method as follows: ΔCt = Ct gene of interest - mean Ct (GAPDH). The fold increase was calculated as 2-ΔΔCt.
Western Blot
Total protein was extracted using RIPA buffer with a total protease phosphatase inhibitor mix (Solarbio, Beijing, China). Samples (60 μg protein) were separated on 10% SDS–PAGE gels and transferred to a polyvinylidene fluoride membrane, blocked with 5% nonfat dried milk in TBST (10 mmol/L Tris, pH 7.5; 150 mmol/L NaCl, 0.05% Tween-20), incubated with primary antibodies including anti-Nrf2 (mouse, 1:2000, Santa Cruz Biotechnology, CA, USA), anti-HO-1 (rabbit, 1:1000), anti-Bcl2 (rabbit, 1:1000), anti-BAX (rabbit, 1:1000), and anti-β-tubulin (rabbit, 1:1000) (Proteintech, Wuhan, China) at 4 °C overnight. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies (EpiZyme Biotech, Shanghai, China). The immunoreactive bands were developed using an ECL kit (Thermo Fisher Scientific, Waltham, MA, USA), and exposure was conducted with the Bio–Rad Molecular Imager Gel Doc TM XR+ (Bio–Rad, Hercules, CA, USA). ImageJ software was used to process and analyse the images.
Immunofluorescence staining
The cells were inoculated onto a sliver that was placed in the lower chamber of the Transwell coculture plate. After 48 h of coculture, the plates were deparaffinized in xylene and rehydrated in graded ethanol. After incubation in a 70 °C water bath with citrate repair solution (pH = 6.0), the plates were incubated with 5% goat serum for 2 h and then with primary anti-Nrf2 (1:50, Santa Cruz Biotechnology, CA, USA) overnight at 4 °C. After that, the plates were washed three times with PBS and then incubated with goat anti-mouse IgG-Cy3 (1:100, Bioss, Beijing, China) for 2 h at room temperature. Nuclei were stained with DAPI (Thermo Fisher Scientific, Waltham, MA, USA). The sliver was removed from the plate and placed on the slide, and the sliver was sealed with gum. Fluorescence images were visualized with confocal microscopy (Leica, Germany).
Animal experiment
Six female BALB/c nude mice (6–8 weeks old) were used in this study. Animals were obtained from SPF Biotechnology (Beijing, China) and maintained in the animal facility of the Fourth Medical Center, Chinese PLA General Hospital. Hypertrophic scar tissue sources are described above. All experimental procedures were performed
following the regulations of the Institutional Animal Care and Use Committee. Scar tissues were washed in PBS and divided into multiple small specimens weighing 0.2 g, with all the skin layers retained. After routine disinfection and anaesthesia, four 1 cm incisions were made in the back skin of each mouse with scissors. The scar tissue fragments were implanted subcutaneously after pockets were created by blunt dissection. The wound was sutured with 5–0 nylon thread and left exposed. The sutures will be removed after a week. Two weeks after transplantation, four hypertrophic scar tissue patches on the back of each mouse were treated in four different ways: A group, blank control, no treatment; B group, negative control, 0.2 ml DMEM/F-12; C group, ADSCs group,0.2 ml ADSCs (resuspended in DMEM/F-12, cell concentration is 1×107/ml); D group, positive control, 0.2 ml triamcinolone acetonide (triamcinolone suspended injection, 40 mg/mL, per vial; Ji Da Chemical & Pharmaceutical Co, Kunming, China). Two weeks later, the injection was repeated, and four weeks later, all the tissue pieces were collected and weighed. In the end, six pieces of hypertrophic scar tissue were collected from each group.
Histological Analysis
Samples from each group were bisected and immediately fixed in 10% formalin. Subsequently, the samples were embedded in paraffin sections and routinely stained with haematoxylin-eosin (H&E) and Masson trichrome reagent. Sections were observed and photographed under a microscope (Olympus, Tokyo, Japan).
Nrf2 protein content
After cutting the sample into small pieces with scissors, the remaining steps were the same as described above.
Statistical analysis
All the results were expressed as the mean ± SEM. Comparisons between the two groups were made using unpaired Student’s t test. For more than two groups, one-way ANOVA with S-N-K post hoc test was used. P < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 8.0 software. (*P<0.05, **P<0.01, ***P<0.001)