Ethics statement
All the procedures including animal studies were approved by Southern Medical University Institutional Review Board and the Nanfang Hospital Animal Ethics Committee and conducted in accordance with the ethical standards of the National Health and Medical Research Council (China).
ALE preparation and osmotic pressure measurement
Human adipose tissues were harvested from female patients undergoing abdominal liposuction in the Plastic Surgery Department of Nanfang Hospital, after taking their informed consent. Detailed procedure of ALE preparation is shown in Fig. 1. First, the harvested adipose tissue was allowed to stand for 10 min in ice water. The liquid portion was discarded and adipose tissue layer collected. The obtained adipose tissue was then washed with phosphate-buffered saline (PBS) and centrifuged at 1200g for 3 min to remove the remaining blood constituents. The concentrated adipose tissue was mixed with an equal volume of PBS and then physically emulsified by repeated transfer between two 10-mL syringes connected by a female-to-female Luer-Lock connector with an internal diameter of 1.4 mm. Transfer of the adipose tissue was carried out for 1, 3, 5, 7, or 10 min at a constant rate (10 mL/s). After centrifugation at 2000g for 5 min, the liquid portion (ALE) from emulsified adipose tissue was collected and passed through a 0.20-μm syringe filter (Xiboshi, DIONEX, USA) to remove cell and tissue debris. The osmotic pressure of ALE was measured using Fiske® 210 Micro Osmometer and the sample stored at − 40 °C until use. ALE emulsified for 1 and 10 min were subjected to mass spectrometric analysis to investigate the effect of time variations on protein yield of ALE. All the five ALE samples were used for cell viability assay and osmotic pressure measurements, while only the ALE emulsified for 1 min was used for all the remaining experiments.
Mass spectrometry identification and database searches
The ALE samples were first digested by trypsin for subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis as described before [25]. The tryptic peptide products were then separated by reverse-phase liquid chromatography using nano LC system (DIONEX Thermo Scientific). Label-free LC-MS/MS identification was subsequently conducted using a Q Exactive Plus (Thermo Fisher Scientific, Marietta, OH, USA) equipped with a self-packed column (Thermo Fisher Scientific, Acclaim PepMap RSLC 50 μm × 15 cm, nano viper, P/N164943) at a flow rate of 300 nL/min according to previous study [26]. Label-free experiments’ data was analyzed using MaxQuant program with high-resolution instruments supported by Andromeda as a database search engine for peptide identification [27]. Label-free quantitation (LFQ) was performed as previously described [28]. LFQ intensity values were used for protein quantification, and protein information was searched against the human database (Uniprot_HomoSapiens_161584_20180123).
Gene Ontology (GO) analysis of ALE was performed to classify all identified proteins into two categories (biological process and molecular function) using Blast2GO v.2 software [29] and web database (www.geneontology.org). Functional annotation of proteins related to angiogenesis and adipogenesis was identified. Ingenuity Pathway Analysis (IPA) analysis of identified proteins in ALE was performed to obtain protein location information through a web database (https://www.qiagenbioinformatics.com).
Growth factor measurement
High sensitivity enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) was used to quantify the levels of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), and vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF) in ALE according to the manufacturer’s instructions.
Cell viability assay
Primary human adipose-derived stem cells (ADSCs) were isolated from adipose tissue as described previously [30, 31]. Briefly, fresh adipose tissue was washed and digested in 0.075% collagenase solution for 40 min on a shaker at 37 °C. The digested tissue was centrifuged at 180g for 5 min and then filtered to remove large debris. Next, the cellular pellet (SVF) was re-suspended in an erythrocyte lysis buffer and centrifuged at 180g for another 5 min. Finally, the SVF was cultured in human ADSC complete growth medium (HUXMD-90011, Cyagen, China). Passage 3 ADSCs were used in subsequent experiments.
ADSCs were treated with the five ALE samples to assess the effect of ALE on cell viability. Briefly, the ADSCs (5 × 103 cells/well) were incubated in a 96-well microplate at 37 °C for 24 h to permit cell adhesion and were later treated with each of the five ALE samples or PBS (control group). The relative cell viability present at the 12, 24, and 36 h time points was estimated using a Cell Counting Kit-8 assay kit (CCK-8, Dojindo, Japan) according to the manufacturer’s instructions. Absorbance was measured using a plate reader at 450 nm, and the OD values in different groups were used to analyze cell viability. These experiments were repeated three times.
Angiogenic induction ability of ALE in vitro
Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in endothelial cell medium (1001, ScienCell, USA). Tube formation assays were conducted by treating HUVECs with ALE to assess the angiogenesis-inducing ability of ALE. In brief, frozen Matrigel (356230; Becton, Dickinson & Co., Franklin Lakes, NJ, USA) was thawed overnight at 4 °C, and all experimental equipment were cooled to − 20 °C before use. The Matrigel (diluted 1:1 in PBS) was transferred to a 24-well plate and solidified by incubation at 37 °C and 5% CO2 for 30 min before seeding of the cells. HUVECs (5 × 105 per well) were then seeded on the Matrigel and treated with 2 mL medium (1:1, ALE to endothelial cell basal medium). HUVECs treated with angiogenic factors [20 ng/mL vascular endothelial growth factor (VEGF) and 5 ng/mL basic fibroblast growth factor (bFGF)] served as a positive control, while cells treated with 2 mL medium (1:1, PBS to endothelial cell medium) were used as a negative control. After 3, 9, and 20 h, tube formation of HUVECs was observed and photographed using a Nikon E200 microscope (Nikon Corp., Tokyo, Japan). Angiogenesis is evaluated by counting the number of tubular structures from six fields of each well. Tube formation assays were repeated three times.
Adipogenic induction ability of ALE in vitro
ADSCs were treated with ALE to assess the adipogenic potential of ALE. In brief, ADSCs (1 × 105 per well) were plated on 6-well plates and cultured overnight at 37 °C and 5% CO2. The next day, cells in each well were rinsed and treated with 2 mL medium (1:1, ALE to human ADSC complete growth medium). ADSCs treated with adipogenesis kit (HUXMD-90031, Cyagen Biosciences, Guangzhou, China) served as the positive control, and cells treated with 2 mL medium (1:1, PBS to human ADSC complete growth medium) were used as the negative control. The medium in all the groups was changed every 3 days. Adipogenic differentiation of ADSCs was examined for lipid accumulation by Oil Red O (ORO; Sigma-Aldrich, USA) staining. Cells were photographed using a Nikon E200 microscope and collected for gene expression analysis at days 4, 7, 14, 21, and 28. Adipogenic induction assays were repeated three times.
Therapeutic effect of ALE in vivo
The therapeutic effect of ALE in vivo was evaluated by the application of ALE in a wound healing model of mice. Female C57BL/6 mice, 4- to 6-week-old (n = 20), were purchased from the Southern Medical University Laboratory Animal Center and were maintained in microisolator cages at the Animal Experiment Center of Nanfang Hospital. For wound healing experiments, mice were anesthetized and two circular full-thickness excisional skin wounds of 8-mm radius were made on the dorsum of mice. A silicone ring was then placed around the perimeter of the wound and secured with 6-0 sutures to prevent wound contraction. Wounds were randomly treated either with 200 μL ALE or 200 μL PBS (control group) in each mouse. After the treatments, the wounds were covered with Tegaderm sterile dressing (3 M Healthcare, St Paul, Minn.). Treatments were administered to the wounds every 2 days, and sterile dressings were changed every day. Pictures of the wounds were taken, and the skins around the wounds as well as health skins were harvested for further analysis on days 3, 7, 11, and 14 after surgery. The wound area was quantified using ImageJ software, and the wound healing was expressed as follows: residual wound area/original wound area × 100.
Histological assessment and immunohistochemical staining
Fresh skin samples were fixed in formalin for histological assessment and immunohistochemical staining. Briefly, formalin-fixed samples were embedded and sliced into 5-μm sections. For histological assessment, tissue sections were deparaffinized in xylene, rehydrated through graded alcohol in phosphate-buffered saline (PBS), and then stained with a hematoxylin-eosin (HE) working solution. For immunohistochemical analysis, sections were dewaxed and rehydrated, then incubated in 3% H2O2 to block endogenous peroxidase. Immunohistochemical staining was performed using antibodies against CD31 (1:25, ab28364, Abcam, Cambridge, UK) and perilipin (1:500, GP29; Progen, Heidelberg, Germany), followed by secondary antibody. Angiogenesis and adipogenesis were evaluated by counting the number of brown-labeled vessel-like structures and brown-labeled adipocytes, respectively, from six fields of each slide.
Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
Total RNA in ADSCs was extracted and measured using qRT-PCR. Primers used for adipogenesis-specific genes were from peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT/enhancer-binding protein alpha (CEBP-α), master regulators for the induction of adipogenic differentiation. RNA was isolated from the samples using Trizol and SYBR Green quantitative PCR SuperMix (Thermo Fisher Scientific, Marietta, USA), followed by reverse transcription. The complementary DNA samples were measured using customized TaqMan Array Plates (Thermo Fisher Scientific, Marietta, USA). Relative expressions were calculated by the cycle threshold method using GAPDH as an endogenous reference gene.
Statistical analysis
Data are expressed as mean ± SD. Results were analyzed using SPSS 22.0 software. An independent t test and least significant difference post hoc analysis were used to compare two and three groups at a single time point, respectively, and one-way analysis of variance (ANOVA) was used to compare groups at all time points. OD values from different ALE samples were subjected to one-way ANOVA followed by Tukey’s multiple comparison test to evaluate the cell viability with ALE treatment. Values of p < 0.05 were considered statistically significant.
