 488-conjugated secondary antibodies (Invitrogen, California, USA). CD31-positive blood vessels, Ki67-positive proliferating cells and the percentage area of perilipin-positive adipocytes were calculated. To analyse cell apoptosis, terminal deoxynucleotidyl transferase-mediated d-UTP nick end labelling (TUNEL) staining was performed according to the manufacturer’s instructions, and apoptotic cell numbers were calculated. Five random fields were selected for each sample (n = 3/group). All measurements were performed with Image-Pro Plus software (Media Cybernetics; Silver Spring, MD, USA).
Fat tissue culture and measurement of viable adipocytes
The lipoaspirate was divided into four groups as above: the PBS group, the FELow group, the FEHigh group and the nanofat group. Fat tissue for each group was cultured in an incubator under hypoxic (1% oxygen) conditions. The fat tissue was then examined by immunostaining with perilipin to detect viable adipocytes at the beginning of tissue culture (day 0) and 24 h later (day 1).
Cell culture
For primary culture of ADSCs, the lipoaspirate was digested with 0.3% collagenase at 37 °C for 1 h and then centrifuged at 1500 rpm for 5 min. The cell pellet was resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotic (Gibco, Carlsbad, CA, USA) and then seeded in a cultured dish. Cell passaging was performed until the cells reached 80–90% confluence. Upon reaching passage 2, FE at different concentrations (0, 1, 2 and 5%) was added to the cell culture supplements to evaluate the effects of FE on ADSCs. Cell numbers were counted at each passage to evaluate the pro-proliferative effect of FE on ADSCs. Characterization of ADSCs was performed when the cells reached passage 5.
Human vascular endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin antibiotic. The culture medium was changed every 2 to 3 days, and the culture was maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2.
Characterization of ADSCs
Trilineage differentiation of ADSCs
The trilineage differentiation potential of ADSCs was measured as previously described [23]. All chemicals were purchased from Sigma (St. Louis, MO, USA) unless otherwise stated. For adipogenic differentiation, ADSCs were cultured in adipogenic induction medium (penicillin/streptomycin, glutamine, 10% FBS, insulin, dexamethasone, 3-isobutyl-methylxanthine and rosiglitazone) for 21 days, followed by Oil Red O staining. For osteogenic differentiation, ADSCs were cultured for 21 days in an osteogenic induction medium (penicillin/streptomycin, glutamine, 10% FBS, dexamethasone, β-glycerophosphate and ascorbic acid) and then assayed by Alizarin Red staining. For chondrogenic differentiation, 5 × 105 ADSCs were pelleted by centrifugation in a 15-ml culture tube. The pelleted ADSCs were then cultured in chondrogenic induction medium using Chondro BulletKit (Lonza Walkersville Inc., Walkersville, MD, USA). After 21 days, the pellet was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned and stained with Alcian Blue solution.
Phenotypic profile of ADSCs
The phenotypic profile of ADSCs was determined by flow cytometry as previously described [24, 25]. Briefly, the cells were collected; stained with phycoerythrin (PE)-conjugated anti-CD11b, anti-CD19, anti-CD34, anti-CD45, anti-HLA-DR, anti-CD73, anti-CD90 and anti-CD105 antibodies (BD Bioscience, San Jose, CA); and analysed using an Epics Altra flow cytometer (Beckman Coulter, CA, USA).
Cell proliferation assay
HUVECs were seeded in a 96-well plate at 1 × 103 cells per well and cultured in DMEM medium containing 10% FBS for 24 h. The cells were then treated for 3 days with different concentrations of FE (0, 1, 2 and 5%). Untreated cells were used as a control. Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Rockville, MD, USA) was used for the cell proliferation assay. The absorbance spectrum at 450 nm was recorded using a microplate reader (SpectraMAX i3x; Molecular Devices, Sunnyvale, CA, USA).
Cell migration assay
HUVECs were seeded in 6-well plates and grown to a monolayer. Wounds were created by scratching with a sterile pipette tip, and the medium was replaced with DMEM supplemented with 2% FBS. FE (0, 1, 2, 5%) was added to the medium. Images were captured with a digital camera at 0 and 24 h after scratching and measured with ImageJ software (NIH, Bethesda, MD, USA). The data are reported as the relative percentage of wounds healed.
Tube formation assay
A tube formation assay was performed in Matrigel (BD Biosciences). HUVECs were suspended in DMEM supplemented with 2% FBS. HUVECs (1.5 × 104) were seeded onto Matrigel-coated 96-well plates. The plate was incubated at 37 °C in 5% CO2 for 6 h. The cells were then stained with Calcein-AM solution (Yeason, Shanghai, China). Tube formation was photographed under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). The number of junctions was calculated using ImageJ software (NIH).
Proteinase K or RNase treatment to investigate the proangiogenic activity of FE
To investigate whether the protein or ribonucleic acid (RNA) component is essential for the proangiogenic effect of FE, FE was digested with proteinase K (PK) (20 μg/ml) or ribonuclease (RNase) (200 μg/ml) at 37 °C for 1 h and then incubated with HUVECs at a concentration of 5%. Cell proliferation assays, migration assays and tube formation assays were performed as described above.
Statistical analysis
Data are presented as the mean ± standard deviation. Differences between the groups were analysed using one-way analysis of variance (ANOVA) or non-parametric tests. Statistical analyses were performed using IBM SPSS software (SPSS Statistics V22, IBM Corporation, USA). Values of p < 0.05 were considered statistically significant.
Results
FE and nanofat improved fat graft survival
To determine the effects of FE on fat transplantation, human fat tissues were transplanted into nude mice together with PBS, nanofat or FE (Fig. 1a). The fat grafts in each group were harvested at 2, 4 and 12 weeks post-transplantation (n = 6/time point/group). Representative grafts in each group are shown in Fig. 1b. A decrease in graft size from 2 to 12 weeks was observed in all groups, which was confirmed by measuring the weight of each fat graft (Fig. 1c–e). The weights of the fat grafts in the nanofat group and the FE-treated groups were significantly (p < 0.05) higher than those in the PBS group. In addition, the weights of the fat grafts in the nanofat group were significantly (p < 0.05) higher than those in the FE-treated groups. No significant difference was observed between the FELow and FEHigh groups.
Histological evaluation of grafts
Histological evaluation of grafts. a HE staining of the harvested fat grafts at 12 weeks post-transplantation. Scale bars = 100 μm. b Histologic analysis of the grafts at 12 weeks post-transplantation. The percentage areas of integrity, vacuoles and fibrosis were calculated (*p < 0.05). c Perilipin staining of the harvested fat grafts at 12 weeks post-transplantation. Scale bars = 50 μm. d The percentage area of viable adipocytes in the grafts at 12 weeks post-transplantation was calculated (*p < 0.05)
Proangiogenic, anti-apoptotic and pro-proliferative effects of FE in vivo
Proangiogenic, anti-apoptotic and pro-proliferative effects of FE on fat grafts. a Anti-CD31 staining of grafts at 12 weeks post-transplantation. Scale bars = 50 μm. b The number of CD31-positive vessels per field in the grafts (*p < 0.05). c TUNEL analysis of grafts harvested at 2 weeks post-transplantation. Scale bars = 50 μm. d The number of apoptotic cells per field in the grafts (*p < 0.05). e Anti-Ki67 staining of grafts at 2 weeks post-transplantation. Scale bars = 50 μm. f The number of Ki67-positive cells per field in the grafts (*p < 0.05)
TUNEL staining showed that the average number of apoptotic cells per field in the PBS, the FELow, the FEHigh and the nanofat groups were 27.25 ± 4.53, 18.89 ± 6.08, 12.52 ± 4.64 and 10.96 ± 5.20, respectively. There were significantly fewer apoptotic cells in the FE and nanofat groups than in the PBS group (Fig. 3c, d), indicating that FE and nanofat might improve graft survival through their anti-apoptotic activity.
The proliferating cells in the grafts were analysed by anti-Ki67 staining. As shown in Fig. 3