Cell Isolation, cultivation and identification of hAMSCs
The placental amniotic tissue was collected from normal pregnant women undergoing full-term cesarean section after gaining the informed consent of the pregnant women or her relatives. This project was approved by the medical ethics committee of Zunyi Medical University (Approval No. 2018. 246). After removing residual blood under sterile conditions, the amniotic tissue was cut into small pieces of 1–2 cm2 and then separated and repeatedly rinsed using phosphate-buffered saline (PBS) containing 100 μg/ml streptomycin and 100 U/ml penicillin (Beyotime, China). Amnion fragments were collected in a 50 mL centrifuge tube and digested for 35 minutes at 37°C in a double volume of digestion solution I (0.05 % trypsin containing 0.02% EDTA-2Na). After centrifugation at 180 rpm for 2 hours at 37°C, the remaining amnion in the centrifuge tube was filtered through a filter screen and digested by a double volume of digestion solution II (0.75 mg/mL collagenase II containing 0.075 mg/mL DNase I) to acquire hAMSCs. The sample was shaken at a speed of 180r/min and digested for approximately 2 hours at 37°C. After filter and centrifuge, the resultant hAMSCs were transferred to low-glucose (LG)-DMEM complete medium, supplemented with 10 percent heat-inactivated fetal bovine serum (FBS) to rest the cell suspension in a humid atmosphere of 5% CO2 for 5 minutes at 37°C to precipitate hAMSC (passage 0, P0).
The resultant hAMSCs (passage 0, P0) were collected in a 50 mL centrifuge tube and digested for 3 minutes at 37°C in a double volume of digestion solution I (0.05 % trypsin containing 0.02% EDTA-2Na). When the cells reached 80% confluence, they were digested and subcultured into flasks. Cells from passage 3 (P3) were used for further analysis in this study. The cell morphology was constantly observed under a light microscope (Olympus, Tokyo, Japan). In addition, these hAMSCs were analyzed using the immunocytochemical staining method according to the protocols as described in a previous study .
For the phenotypic characterization of hAMSCs, P3 hAMSCs were trypsinized and subsequently washed with D-PBS containing 0.1% BSA, adjusted to a density of 1.5×106 cells/mL, and then incubated with and HLA-DR for 25 minutes in the dark. After washing again with D-PBS containing 0.1% BSA, the cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. Finally, the labeled cells were analyzed using the flow cytometry system (FACS Calibur, Becton Dickinson, USA) and CellQuest software (BD, NJ, USA) after fixation with 1% paraformaldehyde.
Cell proliferation assay
This experiment was divided into three groups: blank group (no cells and no ICA), control group (with cells but no ICA) and ICA group (with cells and ICA). The P3 hAMSCs were seeded at a density of 1×104 cells/ml in 96-well plates. After incubation for 24 hours, the medium was changed to ICA-containing media at a concentration of 0 (blank and control), 1×10-4 (ICA-1), 1×10-5 (ICA-2), 1×10-6 (ICA-3), 1×10-7 (ICA-4) and 1×10-8 (ICA-5) mol/L accordingly. Cells were incubated at 37°C in a 5% CO2 incubator for 72 hours, and 20 μL 5 mg/mL MTT was then added to each well, followed by incubation at 37°C in a 5% CO2 incubator for 4 hours. Later, the medium was discarded, and 150 μL of dimethylsulfoxide (DMSO) was added to each well. After incubate at 37°C for 15 minutes, the OD value of each well was determined at a wavelength of 490 nm by a microplate reader (MultiskanTM GO, ThermoFisher, Waltham, MA, USA).
Osteogenic differentiation of hAMSCs in vitro
The P3 hAMSCs were seeded at a density of 1×105 cells/ml in 96-well plates in LG-DMEM culture medium under sterile conditions. After 24 hours of incubation, the medium was replaced with LG-DMEM culture medium with 10% FBS. The experiment was divided into 5 groups according to the different substances added to the medium, namely the classic group (100nmol/L dexamethasone (Sigma, SL, USA), 50 mg/L vitamin C (Solarbio, Beijing, China), and 10mmol/L β-glycerophosphate (Solarbio, Beijing, China)), the blank group (without additives), ICA-1 group (1×10-4 mol/L ICA), ICA-2 group (1×10-5 mol/L ICA), ICA-3 group (1×10-6 mol/L ICA). Cells were incubated at 37°C in a 5% CO2 incubator for 72 hours, and 20 μL 5 mg/mL MTT was then added to each well, followed by incubation at 37°C in a 5% CO2 incubator for 4 hours. The culture medium was changed every 3 days, and the intervention was continuously induced for 21 days. Meanwhile, during the induction period, the cell morphology of the five periods of 1 d, 3 d, 7 d, 14 d, 21 d were photographed for morphological observation, and samples were preserved. ALP (alkaline phosphatase) was extracted and detected with the ALP assay kit (AnaSpec, San Jose, CA) as directed by the manufacturer. The cells on the 21st day were stained with Alizarin red staining (ARS), and statistical analysis was performed based on the number of stained calcium nodules.
Experimental data were expressed as mean±standard deviation (SD) and analyzed using SPSS 19.0 statistical software. One-way analysis of variance was used for comparison between groups, and the rate comparison was performed by χ2 test. P < 0.05 was considered as statistically significant.