Human gingival samples were collected from discarded tissues of third molar extractions at the Qingdao Municipal Hospital. All healthy donors were aged between 18-25 years and provided signed informed consent. All procedures went performed according to the ethical standards and were approved by the Institutional Review Board of Qingdao Municipal Hospital. GMSCs were isolated as described in a previously study . GMSCs obtained by the limited dilution method were cultured in a Minimum Essentia Medium (α-MEM)（Hyclone laboratories, Logan, UT) containing 10% fetal bovine serum (FBS) (Hyclone laboratories, Logan, UT). The cells were sub-cultured at 80% confluence using 0.25% trypsin/EDTA solution （Hyclone laboratories, Logan, UT). Cells (passages 3-6) were used for the following experiments. In addition, Jurkat T cells were provided by the microbiology laboratory of Qingdao university. Murine calvarial cell line (MC3T3-E1) cells were purchased from Gefan (Shanghai, China).
Immunophenotype and differentiation capacity of GMSCs
The immunophenotypic characterization of GMSCs was performed as described in a previous study . The surface antigen expression of the molecules CD34, CD73, CD105, and CD14 (BioLegend, San Diego, CA, USA), was analyzed by flow cytometry (Beckman Coulter, Brea, CA, USA).
To evaluate the multidirectional differentiation of GMSCs. For adipocyte differentiation, GMSCs (5×105 cells/well) were incubated in 6-well plates in adipogenic medium (α-MEM containing 10% FBS, 0.1 μM dexamethasone, 60 μM indomethacin，and 50 mg/ml ascorbate-2-phosphate; Sigma-Aldrich, St. Louis, Mo, USA). The medium was changed every three days. After 14 days, the plates were staining with Oil Red O solution. For osteoblast differentiation, GMSCs (5×105 cells/well) were incubated in 6-well plates in osteogenic medium (α-MEM containing 5% FBS, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and 50 mg/ml ascorbate-2-phosphate; Sigma-Aldrich, St. Louis, Mo, USA). After 28 days, the mineralized nodules were stained with 2% Alizarin red（Solarbio, Beijing, China）.
The colony forming units-fibroblast (CFU-F) assay was performed to evaluate the colony forming efficiency of GMSCs. Five hundred cells were seeded in a 60mm culture dish and cultured for 14 days at 37 ℃ in 5% CO2. After 14 days, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Solarbio, Beijing, China）.
Activation of T-cell
A 50μl antibody solution of anti-CD3 (5ug/ml; Bioss, Beijing, China) was dispensed into each microwell of the 96-well assay plate and kept at 4℃ overnight to coat the culture plates with antibodies. Before adding the T-cell, the antibody solution was decanted and each microwell was rinsed. Soluble anti-CD28mAb (5μg/ml; Bioss, Beijing, China) was added to cells at 2 μg/mL. Cells were incubated for two days, and harvested for subsequent experiments. To determine the optimal culture time, unactivated cells were used as controls. Optical density at 450 nm, that is OD450 of both the groups was detected using Cell Counting kit-8 (CCK-8; Beyotime, Shanghai, China), following manufacturer’s instructions.
Cell proliferation of Jurkat T cells
The effect of GMSCs and Jurkat T cells co-culture times on Jurkat T cell proliferation was estimated by a stimulation index (SI) assay. GMSCs (5×103 cells/well) were plated in a 96-well plate for direct co-culture. After 24h, confirming that the GMSCs was adherent to the wall, the cells were thoroughly washed twice with phosphate-buffered saline (PBS, HyClone laboratories, Logan, UT) and seeded with activated Jurkat T cells. Jurkat T cells were cultured with GMSCs for 24h, 48h, 72h and 96h. After culture, non-adherent Jurkat T cells were collected and treated with CCK-8. Phosphate-buffered saline was used as a control and each sample had 6 wells. The absorbance at 450nm was measured with a microplate spectrophotometer. The stimulation index (SI) was calculated as follows: OD450 of Jurkat T cells with MSCs - OD450 of PBS control)/ (OD450 of Jurkat T -OD450 of PBS control).
The effect of GMSCs and Jurkat T cells co-culture proportions on Jurkat T cell proliferation was estimated by a stimulation index (SI) assay too. Jurkat T cells were co-cultured with GMSCs for 0:1,0.1:1, 0.5:1 and 1:1 ratio. The method was the same as described above.
Immunomodulation capacity of GMSCs
To confirm the possible effect of GMSCs on the inflammatory process, the mRNA expression levels of pro-inflammatory molecules (IL-1β and TNF-α) and anti-inflammatory factors (IL-10) were measured by real time RT-qPCR. TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA) was used to extract the RNA, which was then immediately reverse transcripted to cDNA using the PrimeScript™ RT reagent Kit (Takara, Dalian, China). The primer sequences used are given in Table1. β-Actin was used as an internal control, and the 2 (-Delta Delta C[T]) ( 2−ΔΔCt)method was used to evaluate gene expression levels. The experiments were repeated three times and RT-qPCR was performed three times for all samples.
Preparation of Co-culture supernatant
GMSCs (5×103 cells/well) were plated in a 6-well plate for 24h, and subsequently, activated Jurkat T cells were added. As a control, activated Jurkat T cells were incubated alone in the same medium. After 1 and 3 days, the Jurkat T cells were collected for osteogenic analysis and the supernatants were collected and stored at -20℃ for the following experiments.
MC3T3-E1 cells (2.0×105 cells/well）were seeded in a 6-well plate. Based on the different contents of medium, three groups were considered, which include the supernatants of GMSCs and Jurkat T cells co-culture, Jurkat T cells culture alone and the normal medium. After 3 and 7 days, the cells were collected for analysis.
Alkaline Phosphatase and Alizarin red staining
MC3T3-E1 cells (2.0×105 cells/well) were plated in a 12-well plate for 24h. After adherent growth to 70-80%, the culture medium was removed, and the supernatants of GMSCs and Jurkat T cells co-culture, Jurkat T cells culture alone and the normal medium were respectively added to the cells. On days 3 and 7, cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min at 4°C, and incubated with a mixture of naphthol AS-MX phosphate and fast blue BB salt (ALP Kit, Hongqiao, Shanghai, China). Areas stained as purple were designated as positive. Alizarin red (AL) staining was performed on days 7 and 14. Cell layers were washed with PBS and fixed in 95% alcohol for 15 minutes at room temperature, and later incubated with the AL solution (1%, pH 4.2, Merck Chemicals Ltd, Shanghai, China) for 30 min at 37°C. After aspiration of the overflow, the cells were washed three times with PBS and observed using a microscope (Leica, Hamburg, Germany). All experiments were performed in triplicate.
After 3 and 7 days of culture, the expression of osteopontin (OPN) and COL-1 in MC3T3-E1 cells was detected by immunofluorescence. Adherent cells were fixed in 4% paraformaldehyde at 4 °C, and frozen sections were then permeabilized with 0.5% Triton X-100 for 20 min at room temperature. They were washed with PBS, and incubated with primary antibodies against OPN and COL-1 overnight, followed by fluorescence-tagged secondary antibodies against IgG. The cell nuclei were stained with DAPI (Invitrogen, Carlsbad, CA, USA) before observation with a confocal laser scanning microscope (CLSM, Leica).
Real-time quantitative PCR
After 3 and 7 days in culture, the expression of osteogenic genes were measured, and the mRNA expression of ALP, COL1, and RUNX2 was analyzed using real time RT-qPCR. The primers΄ sequences are displayed as follows (Table2). The method was the same as described in 2.4.
All measurements were conducted at least in triplicate, and all quantitative data are presented as means ± standard deviations. One-way analysis of variance (ANOVA) with Tukey’s test for multiple comparisons was selected for statistical analysis using the SPSS 20.0 statistical software package. The levels of significance were determined at ∗P < 0.05, ∗∗P < 0.01.