The LED irradiation had a wavelength of 860 nm and an optical power output of 60 mA. The radiation source was attached to a support, kept 1 cm from the culture plates. The irradiation was applied for 20 min and 60 min, administering 0.25 and 0.75 J/cm2 of energy intensity.
The PDL cells and OCCM-30 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Caisson Laboratories, North Logan, UT, USA) containing 10% fetal bovine serum (FBS, GeneDireX, Las Vegas, NV, USA) and 100 U/mL penicillin/100 mg/mL streptomycin (PS, Caisson Laboratories) at 37°C in a humidified atmosphere of 5% CO2 in air. The culture medium was changed every 3 days. Cells (5 x 104 cells/mL) were seeded on cover glasses for 24 hours, and transferred to six-well plates in a CO2-independent medium supplemented with 10% FBS and 1% PS. Prior to cell experiments, the cover glasses were sterilized by immersion in 75% ethanol, followed by exposure to ultraviolet light for 6 hours.
The compression and LED application
To apply compression, the flexible plates (Cell were cultured in hydrogels in Bio Press™ Compression Plates) were subjected to static waves with various elongation (0–10%) for 1 hr. using a computer-controlled vacuum stretch apparatus (FX-5000 Compression System, FlexCell International Cooperation). Uses positive pressure to compress samples between a piston and stationary platen on the BioPress™ culture plate yielding up to 2.5kPa of force. After compression, the LED were applied on the plates and cultured for one day, three days and five days. For the control experiments, cells were cultured in the same plates with mechanical force but no LED application. At various times after the force application, cells were processed for analyses of proliferation.
After the various predetermined culture times, cell proliferation was evaluated using the PrestoBlue assay (Invitrogen, Grand Island, NY, USA). Briefly, at the end of the appointed time, the culture plates were harvested and washed with PBS three times. Each plate was filled with 400 mL solution (PrestoBlue: DMEM Z 1:9) and incubated at 37°C for 30 minutes. Plates were read using a multiwell spectrophotometer (Hitachi, Tokyo, Japan) at 570 nm, with a reference wavelength of 600 nm. The results were obtained in triplicate from three separate experiments for each test.11