2.1 Adipose tissue collection
Subcutaneous abdominal adipose tissues were obtained from 4 healthy female donors aged 20-40 years who underwent abdominal liposuction. The donors all provided informed consent. This study was approved by the Ethics Committee of Shanghai Ninth People’s Hospital and complied with the principles of the Declaration of Helsinki. The SVFs were obtained by collagenase digestion as previously described [17].
2.2 Magnetic bead sorting and purity assay
Magnetic bead separation kits (Miltenyi Biotec, Germany) were used to sort CD34+ and CD34- cells. Briefly, 1×108 cells were treated with CD34 magnetic beads at 4°C for 30 min, and a suitable amount of phosphate‐buffered saline (PBS) was added for the centrifugation step. CD34+ and CD34- cells were separated by a magnetic bead separation column. The purity of CD34+ (1×106 cells/100 µl) was analyzed by a FACS Aria flow cytometer (Becton-Dickinson, San Jose, CA, USA).
2.3 Immunophenotypic analysis
For flow cytometric analysis, Passage 4 (P4) CD34+ were isolated by magnetic beads and incubated with monoclonal antibodies against CD73 (APC, clone 555479), CD90 (PE, clone 555479), CD105 (PE, clone 555443), HLA-DR (FITC, clone 555441), CD34 (FITC, clone 555822) and CD45 (PE, clone 555446). All antibodies were purchased from BD Biosciences. The cells were subsequently washed with PBS, fixed with 4% formaldehyde and analyzed on a FACS Aria flow cytometer.
2.4 Tube formation assay
Human umbilical vein endothelial cells (HUVECs) were purchased from the ATCC and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin in a 37°C humidified incubator containing 5% CO2. When the density of the HUVECs reached 90%, the cells were digested and passaged with 0.25% trypsin-EDTA (Sigma, St. Louis, MO, USA). Precooled Matrigel (50 μl per well) was added to a 96-well plate and incubated for 30 min. CD34+ cells, CD34- cells and ADSC (P4) were cultured for 24 hours, and the culture medium was collected and centrifuged at 1700 rpm for 5 min, and the supernatant was used as conditioned medium (CM). The HUVECs were adjusted to 6 × 105 cells/ml and centrifuged, and 50 µl of CM (CD34+-CM, CD34--CM and ADSC-CM) was added. The formation of tubes was observed under a microscope every two hours using a Labovert phase contrast microscope (Leitz, Los Angeles, CA, USA), and images were captured with an attached digital camera (Pixera, Santa Clara, CA, USA).
2.5 Animal model
Thirty-six 6- to 8-week-old male nude mice were purchased from Shanghai Laboratory Animal Center (SLAC). The mice were anesthetized by intraperitoneal injection of 10% glutaraldehyde at a dose of 3.5 ml/kg. A skin flap (5 cm × 0.8 cm) with the pedicle located between the ears was created along the long axis of the body on the backs of the mice. The flap was separated from the deep fascia. All blood vessels were electrocoagulated while exposed, and blood vessels were supplied to the pedicle and suture with 5-0 prolene. All mice were randomly divided into four groups: the control group (n=9, each mice was inject 0.1 ml PBS), CD34+ Group (n=9, each mouse was injected with 0.1 ml, 5×106 CD34+ cells/ml), ADSC Group (n=9, each mouse was injected with 0.1 ml, 5×106 ADSCs/ml) and CD34- Group (n=9, each mouse was injected with 0.1 ml, 5×106 CD34- cells/ml). All animal studies were approved by the Animal Research Committee of Shanghai Ninth People's Hospital.
2.6 Flap necrosis rate evaluation
On the 7th day after model establishment (D7), high-quality photographs of the flaps were captured to evaluate the flap necrosis rate with ImageJ software. The percentages of the necrotic areas were calculated as follows: necrotic area/total flap area×100%.
2.7 H&E and Masson’s trichrome staining
Specimens of distal tissue 3-4 from each flap were harvested on D7. The samples were first fixed in 4% paraformaldehyde for 24 hours and embedded in paraffin. Sections with a thickness of 4 μm were prepared and mounted on poly-l-lysine-coated slides for H&E and Masson’s trichrome staining, as previously described [18]. Light microscopic examination (Olympus Corporation, Tokyo, Japan) and analysis were performed on six random fields of three random sections from each tissue specimen.
2.8 Enzyme‐linked immunosorbent assay (ELISA)
The CM of CD34+ cells, CD34- cells and ADSC (P4) was collected, and the supernatants were used. The concentrations of cytokines such as VEGF, bFGF, TGF-β and IL-10 were measured by ELISA kits from R&D Systems (Minneapolis, MN, USA) in accordance with the manufacturer’s instruction in 96-half-well Maxisorp plates. Cytokine binding was developed with 3,3′,5,5′-tetramethylbenzidine (TMB) and stopped with 1 M sulfuric acid. The absorbance was measured at 450 nm on a standard ELISA reader from BioTek (Bad Friedrichshall).
2.9 RNA isolation and real‐time polymerase chain reaction(RT‐PCR)
TRIzol reagent (Invitrogen, Mulgrave, Australia) was used to extract the total RNA. The RNA was reverse transcribed into complementary DNA by using RevertAid reverse transcriptase (Thermo Scientific, Waltham, MA) according to the manufacturer's instructions. RT‐PCR was performed with SYBR Premix EX Taq (Takara, Dalian, China) by using a ViiA 7 (Life Technologies, Carlsbad, CA). The housekeeping gene β-actin was used for normalization.
2.10 Western blot analysis
Total proteins were extracted from cells with radioimmunoprecipitation assay (RIPA) lysis buffer. After determining the protein concentrations by a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA), twenty micrograms total protein were separated by 10% SDS‐PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes were blocked with 5% bovine serum albumin (BSA) and then probed with primary antibodies against VEGF, bFGF and SDF-1 (1:1000; all from Abcam, Cambridge, UK). The blots were then incubated with HRP‐conjugated secondary antibodies and visualized using an enhanced chemiluminescence detection system (Millipore, Bedford, MA). Quantitative analysis of the immunoreactive bands was conducted by ImageJ software.
2.11 Immunofluorescence staining
Skin sections were incubated with primary antibodies against CD31 or CD68 (all from Abcam, Cambridge, United Kingdom) at 4°C overnight and then visualized using Alexa Fluor 555 secondary antibodies. Fluorescence staining was examined using a confocal laser scanning fluorescence microscope (Carl Zeiss, Jena, Germany). Quantitative analyses were performed by average optical (AO) analysis. The positive expression level was directly proportional to the AO value. AO=IOD /AREA, where IOD represents the cumulative optical density and AREA represents the area of the selected region.
2.12 Statistical analysis
The results are presented as the mean ± SD. Statistical differences among groups were assessed using one-way ANOVA and two-tailed Student's t tests. A value of p < 0.05 was considered statistically significant.