Circadian rhythm of clock gene expression in synchronized human dental pulp cells

The circadian system of living beings is based on transcriptional/translational feedback loops involving a set of clock genes. In vitro, circadian oscillation of clock genes expression has been demonstrated in cell lines, by treating cultures with serum-shock or other synchronizing compounds. We examined whether a serum-shock points out circadian expression of clock genes also in primary cultures of human peripheral tissues, such as the dental pulp. Cells from the dental pulp of three healthy third molars, extracted in 18-year-old orthodontic patients, were grown in medium 199 with 10% fetal bovine serum (FBS) and antibiotics. On 4th passage, confluent cultures were treated with 50% FBS for 2 hours. At 4 hour intervals for 28 hours, cells were harvested with Trizol reagent; the extracted total RNA was analysed for expression of per1, bmal1, cry1 clock genes by RT-PCR. Cosinor analysis of the data was performed to evaluate the presence of circadian rhythmicity. Per1 and bmal1 gene expression oscillated in serum-shocked dental pulp cells with statistically significant circadian rhythm; peaks of per1 and bmal1 expression patterns were in antiphase. The results of the study suggest that primary cultured serum-shocked dental pulp cells may provide an in vitro model of synchronized cells to investigate the existence of circadian periodicity in connective tissue cell activity such as the synthesis of extracellular matrix components