Animals and experiment design
SD rats were purchased from the experimental Animal Laboratory of Henan province (SCXK2007-0001). Ten, 8-week old, male SD rats completed this study. Each cage accommodated 3 to 4 rats. The condition was control temperature (22±2˚C) and light (12:12 h light-dark cycle).
These SD rats were divided into the Con group and Peri group randomly. The Con group just received a standard diet, and the Peri group received standard diet with placing ligatures around the maxillary first molar as described previously . After 4 weeks, these ligatures were removed, and all rats were executed under general anesthesia.
Alveolar bone analysis
After the soft tissue was removed, the maxillae samples were performed as described previously .
Alveolar bone loss was measured (in mm) from the cement-enamel junction (CEJ) to the alveolar bone crest (ABC) for each molar .
These adipose tissues were fixed in 4% paraformaldehyde for 48 h at 4˚C. Then they were dehydrated in an ascending series of ethanol solution and finally embedded in paraffin. For IHC analysis, slices were incubated with some primary antibodies, including mouse monoclonal anti-TLR4 antibody (Abcam), mouse monoclonal anti-TNF-a (Abcam) and rabbit monoclonal anti-IL-1β (Abcam). The immune reaction was observed and recorded using a light microscope (Olympus).
Stem cells were collected from the adipose tissue in superficial abdominal region of rats and maintained in low glucose dulbecoo's modifified eagle's medium (DMEM, Hyclone) containing 10% fetal bovine serum (FBS, Hyclone), 100 μ/ml penicillin (Hyclone) and 100 mg/ml streptomycin (Hyclone). The primary ASCs were cultured for about 7–10 days and then passaged in 2–3 days. The third passage of ASCs was used for immunofluorescence staining and flow cytometry. The characteristics of membrane receptor phenotyping and differentiation assays were used to identify ASCs as reported previously . The markers of ASCs were detected through flow cytometric analysis for CD90 FITC; CD105Percp-CY5.5, CD73 APC, CD31 PE, CD33 PE, CD3 PE (Biosciences). These differentiation abilities of ASCs were assessed by using osteogenesis and adipogenesis inducition. Osteogenic differentiation culture medium was made by low glucose d-MEM medium supplemented with 0.1μM dexamethasone (Sigma), 50μM ascorbic acid (Sigma), 5mM β-glycerophosphate (Sigma) for 4 weeks and finally using Alizarin Red staining to detect the osteocyte calcium deposit. Adipogenic differentiation culture medium was made by high glucose d-MEM medium supplemented with 1μM dexamethasone (Sigma), 0.5M misobutylmethylxanthine (Sigma), 10μg/ml insulin (Sigma), and 100μM indomethacin (Sigma) for 2 weeks, and then determines the adipocyte lipid through Oil Red staining.
CCK-8 Cell Viability Assay
CCK-8 kit (Beyotime) was used to measure ASC's proliferation according to manufacturer’s instructions. To analyze the growth kinetics of ASCs, cells were seeded into 96-well plates (Corning) at a density of 2.5×103 cells/
well. Each well was added 10 ul solution of CCK-8. Plates were incubated in 37˚C, 5% CO2 condition for 2 h. The absorbance values of each well were measured at 470 nm.
Wound healing assay
Cells were seeded at a concentration of 1x105 cells per well in 6-well plates. The culture medium was removed after 18 hours, and a wound was made in the center of each well by scratching with a 200 µl pipette tip. Then, cells were washed twice with PBS and cultured with 2.5% FBS. Scratch wounds were imaged using an inverted microscope (Nikon) at beginning, 24 h, 72 h, and 120 h post-wounding. The average wound widths were analyzed using Image Pro Plus 6.0 software as previously .
Alkaline phosphatase activity
ALP activity was measured using ALP assay kit (NanjingJiancheng Bioengineering Institute) according to the manufacturer’s protocols. To evaluate osteogenic differentiation of these stem cells, cells were seeded into 24-well plates (Costar) at a concentration of 1×104 cells/well, and then incubated in the osteogenesis medium. All data were normalized to total protein content. More than three parallel replicates were analyzed in each group.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using Trizol Reagent kit (Invitrogen) according to the manufacturer’s protocol followed by cDNA synthesis and PCR procedures. Quantitative PCR program was set at 94 °C for 30 s; 40 cycles of 95 °C for 5 s, 60°C for 30 s, and 95 °C for 15 s; followed by 60 °C for 1 min. Relative gene level was calculated using the 2-ΔΔCT method and normalized to GAPDH gene. The primers were designed and synthesized by a company (Sangon Biotechnology Co.). Sequences of target primers used are showed in Table 1.
The total of cells proteins was dissolved in RIPA Lysis Buffer (Servicebio) containing PMSF (Servicebio) in 30°C for 30 min and finally centrifuged at 12000 rpm at 4°C for 10 min. The concentration of protein was measured by BCA protein kit (Servicebio) according to instructions. Proteins were transferred to a PVDF membrane (Millipore) for 1 hour at 200 mA. The membranes were incubated in 5 % skim milk for 2 h. Then, the membrane was incubated with primary antibody at 4°C for 3 h. These primary antibodies as follows: ALP (1:3000, Abcam), BMP2 (1:3000, Abcam), Runx2 (1:3000, Abcam), GAPDH (1:5000, Zhengneng) serves as the internal control in these experiments. Assays were repeated three times and the gray value of western blot stripe was measured by Image J.
All data were expressed as mean ± standard error of the mean (SEM) and 95% confidence intervals. All data were analyzed via t-test. Statistical analysis was performed using Graph Pad Primer 7.0. The value of differences P < 0.05 was considered statistically significant.