Real-time quantitative PCR analysis of human dental pulp stem cells

Excised pulp tissue was digested in a solution containing 3 mg/ml collagenase type I (Merck KGaA, Darmstadt, Germany) and 4mg/ml dispase (Merck KGaA, Darmstadt, Germany) for 1 h at 37°C. After digestion, 5 volumes of α-MEM medium containing 10% FCS were added. This solution was centrifuged at 120 × g for 10min, at room temperature. The precipitated material was resuspended in α-MEM medium and filtered through filters with pores of 70 μM. This procedure resulted in a single-cell suspension for in vitro culture. Isolated DPSCs were randomly assigned to the following experimental groups: Group 1–LPS stimulus and laser irradiation; Group 2–LPS stimulus and no laser irradiation; Group 3–Control group, no LPS or laser treatment. The isolated dental pulp stem cells were stimulated to an inflammatory state, using a lipopolysaccharide (LPS) model, and then subjected or not to laser photobiomodulation. Each experiment was statistically evaluated according to the sample distribution. A total of 85 genes related to inflammation and osteogenesis were evaluated regarding their expression by RT-PCR.