Isolation and culture of ADSCs
Male Sprague-Dawley (SD) rats were purchased from the Experimental Animals Centre of Fujian Medical University. All animal experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Fujian Medical University. Rat ADSCs were isolated from the inguinal fat tissue of SD rats (20-24 weeks old, male) according to a previously reported method. In brief, the inguinal fat tissues were isolated and washed with phosphate-buffered saline (PBS; Invitrogen, Carlsbad, CA). Then, the tissues were minced and digested with 0.1% collagenase type I (Sigma-Aldrich, St. Louis, MO) for 1 hour at 37°C. Enzyme activity was neutralized with low-glucose Dulbecco’s modified Eagle medium (LG-DMEM; HyClone, Logan, UT). After centrifugation, the pellet was resuspended in LG-DMEM supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA). The isolated cells were maintained in tissue culture flasks with culture medium at 37°C in a 5% CO2 incubator.
Confirmation of mesenchymal stem cell (MSC) characteristics
Immunophenotyping was performed by flow cytometry. Briefly, ADSCs were trypsinized, centrifuged, and incubated at 4°C for 30 min with fluorescein isothiocyanate (FITC)-labeled anti-CD34, FITC-labeled anti-CD45, FITC-labeled anti-CD90, and FITC-labeled anti-CD29 antibodies (BioLegend, San Diego, CA). The cells were analyzed using a FACSCalibur cytometer (Becton Dickinson, San Diego, CA), and FlowJo software V10 was used to generate histograms. The multilineage differentiation of ADSCs was analyzed after 21 days of induction. For adipogenic induction, ADSCs were cultured with medium containing adipogenic reagents, and the culture medium was replaced every 2-3 days. After 21 days of incubation, oil red O staining was performed to confirm the formation of lipid droplets. ADSCs were also cultured with medium containing osteogenic reagents for osteogenic induction. The presence of calcium nodules was evaluated by Alizarin red staining.
Preparation of PRP
PRP was obtained from blood drawn from SD rats by cardiac puncture using a two-step centrifugation process according to a previously reported method. The whole blood was centrifuged at 160 × g for 20 min to separate the plasma. The plasma fraction was centrifuged again at 400 × g for 15 min to separate the PRP. A thrombin activator (500 U bovine thrombin in 1 ml 10% calcium chloride) was added to activate the PRP.
Transwell migration assay
To examine ADSCs migration induced by different concentrations of PRP, Transwell Permeable Supports (#3422, CORNING, NY, USA) were applied. ADSCs were placed in the upper chamber of a Transwell plate, while different concentrations of PRP were added to the lower chamber. The concentration gradient of PRP was as follows: 0%, 5%, 10%, 20%, 30%, 40%, 80% and 100%. LG-DMEM containing 10% FBS was added as a control. After incubation for 24 hours at 37°C, the migrated ADSCs were fixed with 4% paraformaldehyde and then stained using crystal violet. Finally, the stained cells were photographed under a microscope for counting.
Diabetic wound model and wound healing assessment
Female SD rats were injected with streptozotocin (Sigma-Aldrich) to induce diabetes. The rats that exhibited hyperglycemia within 1 week after injection were confirmed as diabetic rats, and the rats were then anesthetized by intraperitoneal injection of 10% chloral hydrate. The dorsal hair was removed, two circular wounds (diameter, 10 mm) were created on the dorsum of rats, and silicone rings were sutured around the wounds to prevent contracture. A total of 0.4 ml PBS (control), 0.4 ml ADSCs (1 × 106/ml), 0.4 ml PRP, or 0.4 ml ADSCs (1 × 106/ml) + PRP were administered to each wound of the rats from each group. Wound size was measured from photographs 0, 1, 3, 7, 10 and 14 days after the operation. The experimental design is shown in Figure 1.
The wound tissues were fixed in 4% paraformaldehyde overnight at 4°C. After being washed with PBS, they were dehydrated in a graded ethanol series (30-100%) and embedded in paraffin. Sections (4- to 6-mm-thick) were prepared from the paraffin-embedded wound tissues and then stained with hematoxylin and eosin as well as Masson’s trichrome.
Immunohistochemistry and immunofluorescence
The sections were prepared as described above. For immunohistochemistry, sections were deparaffinized in xylene and dehydrated through a graded series of alcohol. High-pressure antigen retrieval was performed with citrate antigen repair solution and then incubated in 3% hydrogen peroxide at room temperature for 20 min. The slices were incubated with primary rabbit polyclonal antibodies against stromal cell-derived factor-1 (SDF-1, 1:200 dilution, Abcam, Cambridge, MA) at 4°C overnight and then incubated with a horseradish peroxidase-labeled secondary antibody at 37°C for 30 min. Next, 3,3'‑diaminobenzidine (DAB) was added at room temperature for 10 min, and the slices were then stained with hematoxylin at room temperature for 2 min. Finally, the slices were gently washed with deionized water, dehydrated in gradient alcohol solutions, mounted with neutral balsam, and observed using an optical microscope. Anti-CD31 and anti-CD34 antibodies (1:200 dilution, Abcam, Cambridge, MA) were used for immunofluorescence. As for immunohistochemistry, the slices were incubated with the abovementioned primary antibodies at 4°C overnight and then incubated with a secondary antibody at 37°C for 1 hour. The slices were finally stained with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA) and observed under a Zeiss LSM 700 confocal fluorescence microscope.
Enzyme-linked immunosorbent assay (ELISA)
To measure the concentration of VEGF in wound tissues, the specimens were ground, trypsinized and centrifuged to prepare protein extracts. The concentration of VEGF was then determined by an ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Briefly, 96-well plates were coated with an VEGF antibody. Two hundred microliters of standard, control, or sample was added to consecutive wells and incubated at room temperature for 2 hours. Each well was then washed 3 times with wash buffer, and 200 μl VEGF conjugate was added to each well. After incubation at room temperature for 2 hours, 200 μl substrate solution was added to each well, and the samples were incubated for 20 min at room temperature. The concentrations were then determined at 450 nm using a microplate reader.
Protein concentration was quantified using the BCA method. Western blotting was carried out according to the standard protocols. Proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane containing transferred proteins was blocked with 5% skim milk in TBS at room temperature for 1 h and with 1:1000 dilutions of anti-p-STAT3 (Abcam, Cambridge, MA) overnight at 4°C. The next day, the membranes were incubated with secondary antibody at a 1:1000 dilution for 2 h at room temperature after washed with TBST for three times. Chemiluminescence detection was performed using the ECL reagent (Bio-Rad Laboratories). The strength of the signal for each protein was determined based on the corresponding band intensity of the scanned image.
Endothelial cell culture and the MTS assay
Rat dermis microvascular endothelial cells were isolated from dermal microvasculature and cultured as previously described. To evaluate the proliferation of viable endothelial cells, a total of 5 × 103 endothelial cells per well were cultured in 96-well plates in 100 μl medium containing ADSCs culture medium (ADSC-CM), 20% PRP, ADSC-CM+20% PRP or 10% FBS as the control. Twenty microliters of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, Promega, Madison, WI) was added to each well. After incubation for 4 hours, absorption values were measured at a wavelength of 490 nm using a microplate reader.
Statistical analysis was performed with IBM SPSS Statistics 25. All data are expressed as the mean ± SEM. One-way analysis of variance for multiple comparisons with Tukey’s post hoc test was used to determine statistical significance, which was defined by a P-value <0.05.