Cell culture and reagents
hPdLCs were isolated from periodontal ligament tissue obtained from wisdom molars extracted for orthodontic reasons in healthy individuals similar to the method described earlier [26,27]. All donors were systematically healthy, aged from 18 to 22 y.o. Periodontal ligament tissue was scraped from the teeth root surface with a scalpel, cut into small pieces and placed into Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), streptomycin (50 µg/ml) and penicillin (100 U/ml) under humidified air atmosphere of 5% CO2 at 37°C. Outgrowing cells were collected and further grown in DMEM medium. hPdLCs between the third and sixth passages were used in the experiments.
Commercially available LPS from P. gingivalis (Invivogene, San Diego, CA, USA), (R)-(+)- Meth-AEA (Tocris Bristol, UK), and human soluble CD14 (Sigma, St. Louis, MO, USA) were used in the present study.
Cell proliferation/viability assay
Cell proliferation/viability was measured by MTT method, as described in our previous study [18]. hPdLCs were seeded in 24 well plates at a density of 2×104 cells in 500μL of DMEM supplemented with 10% FBS. After 24 h, the media were replaced with DMEM supplemented with 1% FBS and containing meth-AEA at concentrations of 0.03, 0.1, 0.3, 1, 3, 10, 30μM. The hPdLCs were treated with different meth-AEA concentrations for 24 h, and untreated cells were used as a control. After treatment with Meth-AEA, 100 µl of MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, Sigma, St. Louis, MO, USA) were added to each well and plates were incubated at 37°C for 2 h. After incubation, media were discarded, 500 µl of dimethylsulfoxide were added into each well to solve formed formazan crystals, and OD550 values were measured on microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cell proliferation/viability experiments were repeated at least three times for each donor with hPdLCs isolated from five different donors.
Effect of meth-AEA on the production of IL-6, IL-8, and MCP-1 by hPdLCs.
The hPdLCs were seeded in 24 well plate at a density of 5×104 cells in 500μL of DMEM supplemented with 10% FBS. After 24 h, the media were replaced with DMEM supplemented with 1% FBS and containing Meth-AEA at concentrations of 0.1, 1, 10 μM. The hPdLCs were treated with different Meth-AEA concentrations for 24 h, and untreated cells were used as a control. In some experiments, hPdLCs treatment was performed in the presence of 1 µg/ml of P. gingivalis LPS and 0.25 µg/ml soluble CD14. As shown by our recent study, sCD14 enhances the response of periodontal ligament cells to bacterial LPS [28] After 24 h stimulation, the expression of IL-6, IL-8, and MCP-1 in hPdLCs was measured by real-time PCR. The content of the corresponding protein in conditioned media was assayed by ELISA similarly to the methods described previously [18,29].
Isolation of mRNA from hPdLCs, subsequent transcription to cDNA, and amplification were performed using commercially available TaqMan Gene Expression Cells-to-CT kit (Ambion/Applied Biosystems, Foster City, CA, USA), which provides excellent accuracy and superior sensitivity of gene-expression analysis [30]. qPCR was performed on an ABI StepOnePlus device (Applied Biosystems, Foster City, CA, USA) in paired reactions using the Taqman gene expression assays with following ID numbers (all from Applied Biosystems, Foster City, CA, USA): IL-6, Hs00985639_m1; IL-8, Hs00174103_m1; MCP-1, Hs00234140_m1; β2-microglobulin, Hs99999907_m1. Real-time PCR reactions were performed in triplicate in 96-well plates using the following thermocycling conditions: 95°C for 10 min; 40 cycles, each for 15 s at 95°C and 60°C for 1 min. The point at which the PCR product was first detected above a fixed threshold (cycle threshold, Ct), was determined for each sample. Changes in the expression of target genes were calculated using the 2-ΔΔCt method [31], where ΔΔCt = (Cttarget-Ctβ2-microglobulin)sample-(Cttarget-Ctβ2-microglobulin)control, taking an untreated sample as a control.
Content of IL-6, IL-8, and MCP-1 proteins in conditioned media was measured by commercially available ELISA Ready-Set-Go kits (eBioscience, San Diego, CA, USA) according to manufacturer’s instruction. Each measurement was performed in duplicates. For measurement of IL-6 and MCP-1, samples were not diluted, whereas, for measurements of IL-8, samples were diluted 1:10.
Statistical analysis
The normal distribution of all data was tested with the Kolmogorov-Smirnov test. After confirming normal distribution, the statistical differences between different groups were analyzed by one-way analysis of variance (ANOVA) for repeated measures followed by t-test. Statistical analyses were performed using the statistical program SPSS 21.0 (SPSS, Chicago, IL, USA). Curve fitting and regression analysis were performed with Microsoft Excel (Redmond, WA, USA). Data are expressed as mean ± S.E.M. of five different donors. Differences were considered to be statistically significant at p < 0.05.