Preparation of BM-DFATs, SC-DFATs, BM-MSCs, and ASCs
Femur bone marrow and subcutaneous adipose tissue were provided by knee osteoarthritis patients who had undergone total knee arthroplasty at Itabashi Hospital, Nihon University School of Medicine, Tokyo, Japan (n = 9, average age 62.2 ± 15.0 years). Informed consent was given before surgery, and all experiments were conducted with the approval of the Nihon University Clinical Research Review Board. Preparation of SC-DFATs was performed according to a previous report by Matsumoto et al. . Briefly, mature adipocytes were incubated by a ceiling culture method in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA) medium containing 20% fetal bovine serum (FBS, JRH Biosciences, Lenexa, KS). ASCs were prepared according to the preparation method of Zuk et al. . Briefly, the adipose tissue was treated with collagenase, centrifuged, and then the sedimented stromal vascular fraction was incubated by adherent culture in DMEM containing 20% FBS. For the preparation of BM-DFATs, bone marrow fluid collected from the femur was centrifuged, and mature adipocytes were isolated from the floating fraction and cultured by the ceiling culture method. BM-MSCs were prepared by the method described previously . Briefly, the bone marrow fluid was centrifuged, and the precipitate fraction was cultured on plastic dishes. These four cell types were used for experiments within passage 3.
The immunophenotypes of the BM-DFATs, SC-DFATs, BM-MSCs, ASCs at passage 2 were identified using flow cytometry as previously described . The cells grown to 60% confluence were suspended at a density of 5 × 105 cells per tube and incubated with various anti-human antibodies conjugated with phycoerythrin (PE) or allophycocyanin (APC). The following antibodies were used: anti-CD73-PE, anti-CD90-APC, anti-CD105-PE, anti-CD31-PE, anti-CD45-APC, anti-HLA-DR-PE, anti-CD106-PE, anti-CD54-APC, and anti-CD36-PE (all from BD Biosciences, San Jose, CA). Mouse IgG1-PE, mouse IgG1-APC, mouse IgG2a-PE, mouse IgG2b-APC, and mouse IgM-PE (all from BD Biosciences) were used as negative controls. The fluorescence intensity of the cells was evaluated by a FACSAria flow cytometer (Becton Dickinson, Bedford, NJ), and data were analyzed using FlowJo software (version 10.6.1, FlowJo, Ashland, OR). Positive cells were counted and compared with the signal of corresponding immunoglobulin isotypes. A minimum of 1 × 104 events were recorded for each sample, and analysis was performed at least three separate times for each condition tested.
Total RNA was extracted from SC-DFATs, BM-DFATs, ASCs, and BM-MSCs at passage 0 and 1 using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The quality of the extracted RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The GeneChip™ 3′IVT PLUS Reagent Kit (Affymetrix, Santa Clara, CA) was used to synthesize and purify the cDNA and for in vitro transcription, T7RNA amplification and labeling. Fragmentation of cRNA was performed with the GeneChip™ Hybridization, Wash, and Stain Kit (Thermo Fisher Scientific, Waltham, MA). The RNA samples were hybridized to probes using GeneChip™ Human Genome U133 Plus 2.0 Array (Affymetrix) according to the manufacturer’s instructions. Fluorescent images were visualized using a GeneChip Scanner 3000 (Affymetrix). Gene expression data were analyzed using Transcriptome Analysis Console software (version 4.0, Affymetrix) following the software guidelines. An adjusted p-value < 0.05 and log-FC ≥ ± 2.0 were set as the cut-off criteria to screen differentially expressed genes (DEGs).
In vitro differentiation assay
The adipogenic and osteogenic differentiation assay was performed as described previously . Briefly, 5 × 104 cells at passage 3 were seeded on 30-mm dishes (BD Falcon, Franklin Lakes, NJ) and cultured in DMEM containing 10% FBS until reaching confluence. For adipogenic differentiation, the cells were cultured in DMEM containing 10% FBS, 1 mM dexamethasone (Sigma-Aldrich, St. Louis, MO), 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), and 1 × insulin-transferrin-selenium-X (ITS; Invitrogen) for one week. The culture medium was changed every three days. After fixing the cells with 4% paraformaldehyde (Wako, Osaka, Japan), they were stained with Oil red O (Sigma-Aldrich) for 15 min. For osteogenic differentiation, the cells were cultured in DMEM containing 10% FBS, 100 nM dexamethasone, 10 mM β-glycerophosphate (Sigma-Aldrich), and 0.05 mM L-ascorbic acid (Sigma-Aldrich) for one week. The culture medium was changed every three days. After fixing the cells with 4% paraformaldehyde, the cells were incubated at 37°C for 1 h with 0.16% naphthol AS-TR phosphate (Sigma-Aldrich) and 0.8% Fast Blue BB (Wako) dissolved in 0.1M Tris buffer (pH 9.0) for detection of alkaline phosphatase (ALP) activity. The cells were also stained with 1% alizarin red S (Sigma-Aldrich) for 3 min at room temperature. The samples were observed under a BX51 microscope (Olympus, Tokyo, Japan).
Real-time reverse transcription-polymerase chain reaction (RT-PCR)
The mRNA expressions of cells were analyzed by real-time RT-PCR using TaqMan™ gene expression assay. Total mRNA was extracted from cells at passage 3 using an RNeasy Mini Kit, and 1 µg total RNA was reverse-transcribed using a High Capacity cDNA Reverse Transcription Kit (Life Technologies) according to the manufacturer’s instructions. Subsequently, 5 ng cDNA was analyzed by real-time RT-PCR using TaqMan™ Fast Advanced Master Mix (Applied Biosystems, Foster City, CA) and a StepOnePlus Real-Time PCR System (Applied Biosystems). TaqMan™ probes (Life Technologies) for specific genes were as follows: PPARG (PPARg), Hs001115513_m1; RUNX2, Hs00231692_m1; CEBPA (C/EBPa), Hs00269972_s1; ALPL (Alkaline phosphatase), Hs01029144_m1; SLC2A4 (GLUT4), Hs00168966_m1; BGLAP (Osteocalcin), Hs00168966_m1. Expression level of transcripts was normalized to endogenous human 18S ribosomal RNA (4319413E) mRNA levels according to the formulae comparative Ct. Each sample was analyzed in triplicate.
Male SCID mice were purchased from Oriental Yeast Co., Ltd., Tokyo, Japan. The mice were bred in cages maintained in an optimal environment without restriction on eating and drinking. The animal experiments were performed with the approval of the Animal Experiment Committee of Nihon University School of Medicine. Animal breeding and experiments were conducted in accordance with the Animal Experiment Guideline of Nihon University School of Medicine.
Mouse femoral fracture model
The mouse femoral fracture model was created according to the report by Bonnarens and Einhorn . Under inhalation anesthesia with isoflurane, a left transverse femoral fracture was created at 10-mm distance from the knee joint using a micro-bone saw (Zimmer Biomet, Warsaw, IN). Then, 50 µl of peptide hydrogel (PHG) Pura Matrix™ (3-D Matrix, Tokyo, Japan) was injected locally into the fracture gap. PHG was prepared according to the manufacturer’s manual. A 25G injection needle (Terumo, Tokyo, Japan) was inserted intramedullary from the distal femur to fix the bone fracture.
Time course experiment in the mouse femoral fracture model
Male 8-week-old SCID mice (n = 6) were used for the experiment. At 4, 6, and 8 weeks after the femoral fracture, mice were euthanized and both femurs were removed. Micro-computed tomography (CT) images of both femurs were obtained with an R mCT system (Rigaku Co., Ltd., Tokyo, Japan) at 90 kV/100 µA to evaluate the morphological changes of the fracture sites.
Cell transplantation experiment in the mouse femoral fracture model
Male 8-week-old SCID mice (n = 50) were divided into five groups, BM-DFATs group, SC-DFATs group, BM-MSCs group, ASCs group, and Control group, and the fracture model was created for the left femur using the above method (n = 10 in each group). After mixing 1 × 105 BM-DFATs, SC-DFATs, BM-MSCs, and ASCs (passage 2) with 50 µl of PHG, the solutions were immediately injected into the fracture gap. In the control group, only 50 µl of PHG was injected into the fracture gap. Four weeks after model creation, all mice were euthanized and bilateral femurs were removed. Micro-CT images of both femurs were taken to evaluate the effects of transplantation of each cell type on fracture healing. Mice that had an oblique fracture when creating the fracture model were excluded (ASCs group: n = 1, Control group: n = 1).
Bone structure analysis
Based on the micro-CT images, bone structure analysis was performed using image analysis software i-viewR (MORITA, Kyoto, Japan). Bone volume (BV) of the femurs was measured in a 4 × 4 × 4 mm3 area at the center of the fracture, and BMD was calculated.
All data are expressed as mean ± standard error (SE). For comparison between groups, a test of significant difference was performed by one-way ANOVA and Tukey-Kramer multiple comparison test. A value of p < 0.05 was considered as statistically significant. GraphPad Prism Ver5.0 (GraphPad Software, La Jolla, CA) was used for statistical analysis.