Patients and ethics approval
All human tissues including HS tissues, adjacent full-thickness normal skin tissues, and adipose tissues were obtained from patients (mean age 30 years) who underwent surgical excision in Xijing Hospital (Xi’an, China). Before surgery, all patients were informed of the purpose and procedures of this study and agreed to offer their excised tissues. Written consent was obtained from all participants involved in this study. All protocols were approved by the Ethics Committee of Xijing Hospital affiliated with Fourth Military Medical University.
Eight- to 10-week-old BALB/c mice were purchased from the Experimental Animal Center of the Fourth Military Medical University. The animal experimental procedures were performed in strict accordance with Experimental Animal Committee of the Fourth Military Medical University (Xi’an, China).
Human-derived HSFs and NSFs were prepared as previously reported . Briefly, the dermal portions were minced and cultured by tissue block explant to isolate HSFs and NSFs. Cells were incubated with Dulbecco’s modified Eagle medium (DMEM; GIBCO, Grand Island, NY, USA) supplemented with 10 % fetal calf serum (FCS; GIBCO), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a 5 % (v/v) CO2 humidified atmosphere. HSFs and NSFs between the third and fifth passages were used for the following experiments. Cells were seeded in six-well plates at a density of 2 × 105 cells per well; 70–80 % confluent HSFs and NSFs were starved for 12–16 h in serum-free medium. The cells were subjected to mRNA and protein analysis to assess the expression of the fibrotic markers (Col1, Col3, and α-SMA).
ADSCs were cultured as previously described . Six different human subcutaneous adipose tissue samples were obtained after liposuction surgery (n = 6). BALB/c mice were sacrificed and subcutaneous adipose tissue samples were obtained (n = 6). Adipose tissues were minced and incubated with 0.1 % collagenase I (Catalog No.17100-017, Life Technologies, GIBCO) in phosphate-buffered saline (PBS) with 2 mM calcium chloride for 1 h at 37 °C. The digested tissues were collected and filtered through a 75-μm mesh, then centrifuged at 200 × g for 5 min, and the supernatant was discarded. Cell precipitates were re-suspended in F12/DMEM (GIBCO) containing 10 % FCS. When the third to fifth passages of ADSCs achieved 80–90 % confluence, cells were starved with serum-free medium for 24 h. Then, the supernatant of ADSCs was collected, centrifuged at 200 × g for 5 min and filtered using a 0.22-μm syringe filter, and finally used as the ADSC-CM. Different concentrations of diluted ADSC-CM (10 %, 20 %, 40 %, 80 %) in mixed medium (F12/DMEM:DMEM) were used in the following experiments; an 80 % concentration of diluted mouse ADSC-CM was prepared for the following experiment.
Flow cytometric analysis
Cell surface marker expression was examined as follows: Fluorochrome-conjugated anti-human CD14-FITC, CD31-FITC, CD34-PE, CD44-PE, CD29-APC, CD73-PE, and CD90-FITC antibodies and Fluorochrome-conjugated anti-mouse CD29-FITC, CD31-FITC, CD44-PE, CD45-PE, and CD106-FITC antibodies were purchased from BD Pharmingen (San Diego, CA, USA) and used in accordance with the instructions of the manufacturer. Non-specific staining was controlled by the use of isotype-matched antibodies. ADSC suspensions were incubated with the primary antibodies (1:50) for 30 min at room temperature. After incubation, the cells were washed twice with PBS and analyzed using a flow cytometer (BD FACSAria™ III system; BD Pharmingen).
Adipogenic and osteogenic differentiation
ADSCs at passages 3–5 were seeded in six-well plates that were pre-coated with a 0.1 % gelatin solution (Cyagen Bioscience, Inc., Guangzhou, China) at a density of 105 cells per well and allowed to reach 80–90 % confluence. Adipogenic differentiation was induced by using a basic medium with 0.5 μmol/l dexamethasone, 0.5 mmol/l 3-isobutyl-1-methylxanthine, 0.1 mmol/l rosiglitazone, and 100 UI insulin for 2 weeks (Cyagen Bioscience, Inc. HUXMD-90031); osteogenic differentiation was achieved by using a basic medium containing 0.1 μmol/l dexamethasone, 50 μmol/l ascorbic acid, and 10 mmol/l β-glycerophosphate for 3 weeks (Cyagen Bioscience, Inc. HUXMD-90021). Medium was replaced every 3 days.
At the endpoint, cells were fixed with 4 % paraformaldehyde in PBS for 15 min at room temperature and stained with specific oil red O and alizarin red S following the manufacturers’ instructions, respectively, to assess adipogenic and osteogenic differentiation. Specific-stained ADSCs were documented under the Olympus IX71 light microscope (Olympus, Tokyo, Japan).
Real-time quantitative polymerase chain reaction (qRT-PCR)
qRT-PCR was performed as previously reported . In brief, total HSF and NSF RNA were extracted after 24 h of incubation with or without different concentrations of diluted ADSC-CM by using a RNA isolation kit (Takara, Dalian, China). RNA purity was evaluated by calculating the A260/A280 ratio, aiming for a value of 1.8–2.1. The primer pairs (human) used for gene amplification from the cDNA template were as follows: Col1: forward 5′-GAGGGCAACAGCAGGTTCACTTA-3′ and reverse 5′-TCAGCACCACCGATGTCCA-3′; Col3: forward 5′-CCACGGAAACACTGGTGGAC-3′ and reverse 5′-GCCAGCTGCACATCAAGGAC-3′; α-SMA: forward 5′-GACAATGGCTCTGGGCTCTGTAA-3′ and reverse 5′-TGTGCTTCGTCACCCACGTA-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH): forward 5′-GCACCGTCAAGCTGAGAAC-3′ and reverse 5′-TGGTGAAGACGCCAGTGGA-3′. The results from three independent reactions were used to determine the relative expression levels of the target genes, which were normalized against the expression level of the GAPDH control.
Western blot analysis
Cells were harvested after 24 h of treatment with different concentrations of diluted ADSC-CM or 1 h of treatment with the p38 inhibitor SB203580 [20, 21] (10 μM Beyotime, Shanghai, China) and/or 30 min of treatment with the p38 activator anisomycin [22, 23] (25 μg/ml, C14H19NO4 cell signaling technology, Inc.), washed in PBS, and re-suspended in RIPA cell lysis solution (Beyotime) supplemented with phenylmethylsulfonyl fluoride (PMSF; Boster, Wuhan, China). The proteins in cultured HS tissues were extracted by RIPA + PMSF with a high-throughput tissue grinder. The protein concentration in each sample was determined by using a BCA Protein Assay Kit (Beyotime). Western blot analysis was performed as previously described . Briefly, cell lysates containing equal amounts of protein (50 μg) were separated in 10 % sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride membranes at 100 V for 40–100 min (Millipore, Billerica, MA, USA). The membranes were blocked with 5 % non-fat milk in tris-buffered saline/0.5 % Tween-20 at room temperature for 3–6 h, and then incubated at 4 °C overnight with anti-Col1 (Rabbit, 1:1000; Abcam, Cambridge, UK), anti-Col3 (Rabbit, 1:3000; Abcam), anti-α-SMA (Rabbit, 1:1000; Abcam), anti-p38MAPK (Rabbit, 1:1000; Cell Signaling, Thermos Fisher Scientific Inc., USA), anti-phospho-p38MAPK (Thr180/Tyr182) (Rabbit, 1:1000; Cell Signaling), and anti-β-actin antibodies (Goat, 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The membranes were then washed four times with tris-buffered saline/0.5 % Tween-20 and incubated at 37 °C for 1 h with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:3000; Boster). The membranes were then washed again four times with tris-buffered saline/0.5 % Tween-20 and immunoreactive traces were detected by using an ECL Kit (Millipore, Billerica, MA, USA). The intensity of protein expression on the membranes was analyzed by Image J software.
Fibroblasts cultured on coverslips were seeded in 12-well plates and fixed in 4 % paraformaldehyde in PBS for 15 min at room temperature, following by washing with PBS, three times per 5 min. Then, fibroblasts were incubated with 0.1 % Triton X-100 in PBS for 30 min, following by washing with PBS, three times per 5 min again. After blocking with1 % bovine serum albumin in PBS for 1 h, the cells were incubated in BSA blocking buffer containing primary antibodies-α-SMA (1:200; Abcam), the cells were washed and then incubated in secondary antibodies anti-rabbit (1:200; Life Technologies, Carlsbad, CA, USA) for 1 h. Furthermore, fibroblasts were incubated for 15 min with 4,6-diamidino-2-phenylindole (DAPI; ZSGB, Beijing, China) for nuclear staining, and fluorescence micrographs were obtained using an Olympus FSX100 microscope.
Cultured HS tissues ex vivo
Human HS tissues that satisfied the following requirements were used in the study: 20–50 years; hypertrophic scars had not faded within a year; the patients did not administer any drugs before surgery; confirmation of clinical diagnosis according to the appearance (thickened, raised (>2 mm), red, hard, itchy, and the relevant pathological diagnosis) (n = 6). Cultured HS tissues ex vivo were performed as described previously [24–26]. Subcutaneous fat tissues were removed, and HS tissues were cut into 10 × 10 mm sections and cultured with DMEM/80 % ADSC-CM in the presence of a p38 inhibitor (SB203580; 10 μM) and activator (anisomycin; 25 μg/ml). Explants containing complete epidermal and dermal layers were cultured in an air liquid interface with the epidermal and keratin layers side up and exposed to air. The medium was changed every 3 days. HS tissues were harvested after 7 days, and were fixed 4 % paraformaldehyde in PBS for Masson’s trichrome staining or extracted for total protein to measure the expression of p-p38, Col1, Col3, and α-SMA.
BABL/c mice excisional model
BALB/c mice were randomly divided into two groups, which were subcutaneously injected with either DMEM or the equivalent mixed medium containing a 80 % concentration of ADSC-CM 1 day before the incision was made (Day –1). Medium (1 ml) was injected subcutaneously at four points in the 1 cm × 1 cm region, with each point receiving approximately the same volume. One day later (Day 0) the skin was prepared with iodine solution. Full-thickness excisional wounds of 1 cm × 1 cm were created using a template on the dorsal skin of 8- to 10-week-old BALB/c mice [27–29]. Three days later (Day 3), experimental models were injected again around the wound with the same volume as described above. The injections were repeated after six days (Day 6). Furthermore, to explore the anti-fibrosis effect of ADSC-CM through the p38/MAPK signaling pathway, we randomly divided BALB/c mice into six groups (n = 6): DMEM, DMEM + SB203580 (5 mg/kg) , DMEM + SB203580 + anisomycin (6.25 mg/kg) , 80 % ADSC-CM, 80 % ADSC-CM + SB203580, 80 % ADSC-CM + SB203580 + anisomycin. Daily photos of the wound were obtained for 2 weeks. The mice were then sacrificed on Day 14 and the skin tissues of the wound were harvested. The specimens were fixed in 4 % paraformaldehyde and sections were used for hematoxylin and eosin (H&E), Masson’s trichrome staining, and immunohistochemistry staining.
Histopathology, immunohistochemistry, and Masson’s trichrome staining analysis
The wound healing samples in vivo or cultured HS tissues ex vivo were embedded in paraffin blocks and cut into 4-μm thick tissue sections. One section was used for routine H&E staining and another was used for Masson’s trichrome staining analysis of collagen fibers. For immunohistochemistry staining, sections were dewaxed and endogenous peroxidase activity was quenched with 3 % hydrogen peroxide for 15 min. The sections were then blocked with normal goat serum for 30 min to eliminate non-specific binding and incubated overnight at 4 °C with primary antibodies against Col1 or Col3 (1:100; Abcam). On the next day, the sections were treated with a PV6000 Histostain™ kit (ZSGB, Beijing, China) and stained with diaminobenzidine (ZSGB, Beijing, China). Finally, the sections were counterstained with hematoxylin. An isotype-matched IgG served as the negative control for each immunostaining procedure. Sections were analyzed and images acquired with FSX100.
All data were analyzed using SPSS17.0 software; every experiment was repeated at least three times, and the data were presented as mean ± standard error of the mean. Statistical analysis was performed by Student’s t tests. P < 0.05 was considered statistically significant.