OBJECTIVETo analyze the effects of calcium hydroxide [Ca(OH)2] on transcription of the bone sialoprotein (BSP) gene in human dental pulp cells.

METHODSHuman dental pulp tissues were collected from extracted teeth for orthodontic reason. In cell culture media, different dose (0.012, 0.120, 0.400 and 1.200 mmol/L) of Ca(OH)2 was added. Total RNA of cells were extracted. The best dose of Ca(OH)2 on human BSP was determined with the real-time polymerase chain reaction (PCR). Further, the time (0, 3, 6, 12, 24 h) effects of the best dose Ca(OH)2 on human BSP, runt-related transcription factor-2 (Runx-2) and osterix (OSX) mRNA levels were determined with PCR. Further method included transient transfection assays, linking chimeric constructs of the human BSP gene promoter to a luciferase reporter gene, then ransfected using lipofectamine in cells and measured the luciferase activities of BSP gene promoter.

RESULTSWith the real-time PCR, the optimal Ca(OH)2 concentration was determined as 1.200 mmol/L. With this concentration at different time points (0, 3, 6, 12 and 24 h), the levels of BSP mRNA increased at 6 h (1.45 ± 0.36), reached maximal at 12 h (2.66 ± 0.18); the levels of Runx-2mRNA increased at 6 h (2.38 ± 0.08), at 12 h (2.73 ± 0.16), and decreased at 24 h. OSX mRNA could be recognized at 12 h, reached maximal levels at 24 h (3.30 ± 0.062). Transient transfection assays showed that treatment of human dental pulp cells with Ca(OH)2 (1.200 mmol/L) increased the luciferase activities of the constructs between -84LUC and -868LUC at 12 h (2.00 ~ 2.60 fold).

CONCLUSIONSThis study demonstrate that Ca(OH)2 could stimulate BSP transcription between -84LUC and -868LUC in the human BSP gene promoter in human dental pulp cells.