Culturing and Identifying of Human Adipose-Derived Stem Cells (hADSC).
We harvested the human subcutaneous fat tissue of healthy donors who had liposuction. The use of human adipose tissue was approved by the Ethics Committee of Plastic Surgery Hospital. The fresh fat specimen was washed three times with PBS containing 1% penicillin/streptomycin. The fat tissue was digested with 0.25% collagenase type I (Sigma, USA) at 37℃ for 0.5h and centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended and filtered through a 70-μm filter (Corning, USA). After another centrifugation for 5 min, the cell pellet was resuspended in the culture medium consisting of Human Mesenchymal Stem Cell Medium (MSCM, ScienCell, USA) high glucose. The culture medium was replaced 48h after seeding to remove nonadherent cells; thereafter, the medium was replenished every 2–3 days. The ASCs were passaged 3 times for experiments. The hADSCs immunophenotype was analyzed by flow cytometry using the flow cytometer FACSCalibur (BD, New Jersey, USA). hADSCs cultivated to passage 3 were labeled with Human MSC Analysis Kit (BD, New Jersey, USA). Cell samples were labeled with each antibody separately and after processing, concentrated cell populations were gated and then the percentage of cells labeled with the selected antibodies was identified, as proposed by the International Federation for Adipose Therapeutics and Science (IFATS) and International Cell Therapy Society(10). The data use isotype as a reference and analysis were performed using the FlowJo7.6.1 Software.
Filler Preparation and Grouping
All animal study protocols and procedures were approved by the Animal Care Ethics Committee of Plastic Surgery Hospital. The animals used in this study included 12 adult male Sprague–Dawley rats weighing 350-450g. Rats were placed under general anesthesia by intraperitoneal injection of sodium pentobarbital (50 mg/kg). The rat was supine with the groin exposed. Open the skin along the groin to reveal a large amount of milky adipose tissue. The adipose tissue was carefully removed and the hemostasis was complete. The surgical wound was closed with 4-0 nylon sutures, and the rat was changed to the prone position. Fillers were then injected into the rat's back (Figure 1A). The whole process is carried out under aseptic conditions.
The adipose tissue was loaded into a 20ml spiral syringe, and cut into pieces with scissors. A small amount of PBS was added during the cutting process for lubrication. Then adipose tissue passe through the 1.0mm aperture converter and loaded into the 15ml centrifuge tube. After centrifugation at 1 000 r/min for 3 min, the upper layer was lipid droplets, the lower layer was PBS, blood and fibrous tissue, and the middle layer was intact fat particles (Figure 1B). The fat particles were loaded into a 15ml centrifuge tube for later use (Figure 1C).
Porcine acellular dermal matrix (PADM) particles (#6846, Unitrump Bio, Qidong, China), autologous fat particles, hADSC and PBS were combined with six different combinations were filled in the rat’s back. They were mixed in the proportions given in Table 1. 18-gauge needles were used to inject each rat subcutaneously at 6 spots, each of the filling material contained a volume of 1.2 ml.
Table 1
Filling ratio
|
A
|
B
|
C
|
D
|
E
|
F
|
Acellular dermal matrix
|
0ml
|
0ml
|
0.4ml
|
0.4ml
|
0.8ml
|
0.8ml
|
Autologous fat particles
|
0.8ml
|
0.8ml
|
0.4ml
|
0.4ml
|
0ml
|
0ml
|
hADSC
|
0ml
|
0.4ml
|
0ml
|
0.4ml
|
0ml
|
0.4ml
|
PBS
|
0.4ml
|
0ml
|
0.4ml
|
0ml
|
0.4ml
|
0ml
|
Assessment of the Filling Material Survival Rates
After 2, 6 and 12 weeks, four rats were selected randomly and euthanized with approved IACUC protocol, and the filling materials were carefully dissected from their back. Each filling material was measured, and the survival ratio for filling material was calculated by using the formula: survival volume/previous volume. Subsequently, each filling material was placed in paraformaldehyde and used for histological and immunohistochemistry examination.
Histological Analysis
The filling material transplants from the 6 groups were embedded in paraffin and cryopreserved in optimal cutting temperature (OCT) media compound. Paraffin-embedded filling material histological sections (5 μm) were prepared and then stained with H&E (PH1732, Phygene, Fuzhou, China). The H&E staining procedure was performed according to standard instructions. The microscope was used to obtain images. Neovascularization was assessed by counting the capillaries in 10 fields of each slide. The images were analyzed by Image Pro Plus software.
Statistical Analysis
All data were presented as the means ± standard error of the mean (SEM). GraphPad Prism 6.0 (USA) was used for statistical analysis and draft graphs. The Student’s t test was used to evaluate the significant differences between the 2 groups. Multigroup comparisons were determined by one-way ANOVA with Bonferroni correction. Multigroup comparisons with different times were analyzed by two-way ANOVA with factors of treatment and time. Intergroup differences were analyzed by performing post hoc Bonferroni tests. P values ≤ 0.05 were considered statistically significant (### or ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05).