Isolation and culture of periodontal ligament cells
The periodontally healthy and non-carious first premolars were collected from young patients without any metabolic and systemic disease referred to the specialized dental clinic of Tehran University of Medical Sciences (TUMS) for orthodontic treatment after receiving informed consent forms. The protocol used in the present study was examined and approved by the Ethics Committee of the Vice Chancellor for Research and Technology of Tehran University of Medical Sciences (IR.TUMS.VCR.REC.1398.480). After being extracted, teeth were immersed in Hank solution containing 1% penicillin / streptomycin and amphotericin B (25 μg / mL) and were rapidly transferred to the Dentistry Research Institute of TUMS.
After rinsing in PBS buffer, periodontal tissue was scraped from the middle third of the tooth root surface followed by 20 minutes incubation in dispase (4 mg/ml) and type 1 collagenase (3 mg / ml) at 37 °C. After 5 min centrifugation (500g), primary cells were transferred to the culture plates and incubated in α-MEM (Cerogen, Darmstadt,Germany) containing 10% fetal bovine serum, 1% penicillin / streptomycin (BioSera, Paris,France), and 1% L-glutamine with 5% CO2 at 37 °C. In this study, cells at fifth to seventh passages were used.
Applying tensile forces
The cells were cultured on a 250 μm-thick Poly DimethylSiloxane (PDMS) (Sylgards184, Dow Corning, Midland, MICH, USA) membrane and examined in a Custom Built machine (Pasteur Institute of Iran, Tehran) under equiaxial tensile stresses as described in detail previously.[14] In order to ensure greater adhesion of cells to silicones surface, PDMS membrane was incubated in 0.2% gelatin for 20 minutes before culturing the cells.
The cells at 80% confluency underwent forces with a frequency of 1 Hz and 10% elongation (strain) for 24, 48, and 72 hours. After each time period, the culture medium was collected and stored at 20°C for ELISA testing.
Applying compressive forces
In the compressive model, cells were cultured on a glass slide (38×25 ×0.2 mm). The silicone membrane of the same size was then placed on glass slides. A total of 25 gr force (25 gr/cm2) was applied on silicone membrane using 240 gr lead granules, each weighing 1 gr. The experiments were performed at intervals of 6, 24, 48, and 72 hours.
Evaluation of gene expression by quantitative real time PCR (qRT-PCR)
RNA was extracted using TRI reagent (Sigma, Roedermark ,Germany). To convert RNA into DNA, 20 μl reaction containing 1 µl random hexamer, 10 µl master mixes and 1000 ng of RNA samples were prepared. The qRT-PCR reaction was performed using 10 µl of 2X Master Mix (Biofact, Daejeon ,Korea). Temperature cycles included 15 minutes at 95 °C, 40 cycles at 95 °C for 15 seconds, 60 °C for 20 seconds, and 70 °C for 20 seconds using System LightCycler® 480 device (Roche, Basel, Switzerland). Table 1 shows the nucleotide sequence of the primers. 2-∆ct was used to calculate the expression level of the targeted genes. The mean expression of Glyceraldehyde-3-Phosphate Dehydrogenase gene (GAPDH) was considered as an internal control to normalize gene expression level.
Evaluation of sclerostin and periostin secretion using ELISA
Human sclerostin and periostin ELISA Kit (Hangzhou EastBiopharm , Hangzhou, China) were used to evaluate the level of sclerostin and periostin in culture medium following the application of tensile and compressive forces. The experiment was performed according to the manufacturer's instructions. The absorbance was read at 450 nm wavelength using microplate reader (BioTek, Winooski, VT, Chittenden ,USA).
Evaluation of cell apoptosis using DAPI (6-diamidino-2-phenylindole) staining
DAPI staining was used to evaluate cell apoptosis after exposing the PDL cells to compressive stresses at 6, 24, 48, and 72 hours. For this aim, cells were fixed with paraformaldehyde 4% for 5 min followed by 3x wash with PBS. Then, cells were permeabilized with 0.2% Triton X100 in PBS for 5 min and incubated with DAPI 1:10000 (Santa Cruz Biotechnology, Dallas, Texas, United States). The cell’s nuclei were examined under fluorescence microscope (Olympus,). The percentage of apoptotic cells with condensed and/or fragmented nuclei were determined by counting >100 cells per field from 5-6 different locations per slide.
Statistical analysis:
All experiments were repeated two times in triplicate. To examine the effect of two variables of time and type of applied forces on gene expression and protein production, two-way ANOVA was used. To examine the significance of time and force type interaction, one-way ANOVA was applied and t-test was used to compare the effect of compressive force with the tensile force applied at the aforementioned time intervals. P-value<0.05 was considered as the significance level.