The dental pulp was washed with PBS containing 1% (v/v) penicillin-streptomycin Y-27632 buy and was placed in 4U of collagenase type 1 at 37��C. Single cells from dental tissues were obtained by pipetting the cells several times in ��-modified Eagle’s medium (AMEM). After this, the cells were centrifuged at 1200g for 10 minutes. The pellet was resuspended with complete medium (AMEM supplemented with 20% v/v fetal bovine serum) and cultured in a 6-well plate. For cultivation, cells were transferred into T25 flasks containing complete medium and kept in incubator with a temperature of 37��C and humidity of 95% and 5% CO2.2.2. Differentiation to Chondrocyte CellsApproximately 1 �� 105cells/mL were transferred to 24-well plates and kept in incubator with a temperature of 37��C and humidity of 95% and 5% CO2.

After washing the cells with 1 X PBS, the cells were placed in chondrogenic medium (Zen-Bio, Inc.) for 21 days to observe for chondrocyte differentiation. Spent medium was replaced with fresh complete medium after every 3 days.2.3. Chondrocyte Cell StainingAfter 21 days of culturing in chondrogenic medium, the cells were first fixed in formaldehyde 4% (v/v) for 2 hours. Then, they were treated sequentially with alcohol 75% (v/v), 95% (v/v), and 100% (v/v). Finally, the cells were stained by toluidine blue for 2 minutes at room temperature. 2.4. RNA Isolation for Chondrocyte CellsTotal RNA was extracted from these cells using Trizol at day 1, 14 and 21. The cells were detached from the T25 flask using 0.25% (v/v) trypsin/EDTA.

Cells were then centrifuged at 1200g for 10 minutes, and the obtained pellet was lysed with 1mL TRIZOL (Regent-Total RNA Isolation Regent) (Invitrogen, USA) for 5 minutes at room temperature. Approximately 0.2mL chloroform (SYSTERM) was added into the sample and vortexed for 15 seconds. This was followed by centrifugation at 12000g for 15 minutes at 4��C. The colorless aqueous phase was taken as the RNA. This phase was transferred into new tube, and 0.5mL of 100% (v/v) isopropanol was added and incubated at room temperature for 10 minutes. After that, the sample was centrifuged at 12000g for 10 minutes at 4��C. The supernatant was discarded, and the RNA pellet was washed with 15% (v/v) ethanol. The sample was centrifuged again at 7500g for 5 minutes at 4��C.

The supernatant was discarded, and the RNA pellet dried at room Dacomitinib temperature for 5 minutes before resuspending in 25mL free nuclease water and incubated at 55��C for 15 minutes. The obtained RNA was stored at -80��C until use.2.5. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)Materials for RT-PCR ( Access Quick RT-PCR Kit System, Promega) were 5X reaction buffer AMV/Tfi, dNTP mix (10mM), forward and reverse primers (50pmol), MgSO4 (25mM), reverse transcriptase (5U/��L), template RNA (0.5��g), and nuclease free water.