Effect Of Simvastatin On The Proliferation And Alkaline Phosphatase Activity Of Human Dental Pulpcell

Objective: To explore the proliferation, the mineralization, the cell cycleand the alkaline phosphatase （ALP） activity of human dental pulp cells（HDPCs） in vitro and the effects of Simvastatin on it.Methods: Healthy, intact and caries-free permanent teeth or third molarswhich had no cracks and were freshly extracted from the people at the age of18to28were provided by the dental surgery of the Second Hospital of theHebei Medical University. Tissue explant-collagenase digestion method wasadopted to culture the cells. When the cells grew out, their morphology andgrowth conditions were observed with an inverted microscope. HDPCs of the4th passage in logarithmic phase were taken to make cellslides.Immunohistochemical staining of vimentin and keratin were used toexamine the origin of cell.The influence of Simvastatin on the proliferation of HDPCs wasmeasured by the MTT assay:1) The dental pulp cells of the4th passage thatgrew well were chosen to prepare the cell suspension of1×10⁴/ml, and theninoculated into96-well plates,100μl per well.2) The culture medium in thewells was thrown away when the cells overgrew the well bottoms, then theplates were washed with PBS three times3) The cells were divided into3groups: control group, blank control group and Simvastatin-treatment group,each group of5wells. Control group: HDPCs were cultured with150μlcomplete medium （high glucose DMEM, including2%fetal bovine serum）.Blank control group:150μl complete medium was added into each well whichhad no cells. Simvastatin-treatment group: HDPCs were stimulated bySimvastatin at different concentrations (10^-9、10^-8、10^-7 and10^-6mol/L)4)The culture medium was thrown away and the plates were washed with PBSthree times when the cells were cultured at different time（1d and5d）. And then the HDPCs continued to be cultured with20μl MTT solution （5g/L）for4h at37℃. The supernatant in the wells was abandoned. Then the plateswere shaken softly at room temperature for5min with100μl DMSO. Theoptical density of every well was measured by ELIASA at a wavelength of492nm. Finally, the average optical density was calculated and compared withthe control group’s.The detection of the influence of Simvastatin on the alkaline phosphatase（ALP） activity of HDPCs:1) The HDPCs of the4th passage that grew wellwere chosen to prepare the cell suspension of1×10⁴/ml, and then inoculatedinto96-well plates,100μl per well.2) The culture medium in the wells wasthrown away when the cells overgrew the well bottoms, then the plates werewashed with PBS three times.3)The HDPCs were divided into3groups:control group, blank control group and Simvastatin-treatment group, eachgroup of5wells. Control group: cells were cultured with150μl completemedium （high glucose DMEM, including2%fetal bovine serum absorbed byactive carbon）. Blank control group:150μl complete medium was added intoeach well which had no cells. Simvastatin-treatment group: HDPCs werestimulated by Simvastatin at different concentrations (10^-9、10^-8、10^-7 and10-6mol/L)4). The culture medium was thrown away and the plates were washedwith PBS three times when the cells were cultured at different time（1d and7d）. Then50μl（1g/L）Triton X-100was added into the wells to incubate thecells overnight at4℃. When the cells completed their division, they wereincubated at37℃with100μl ALP substrate mixture. Sodium hydroxide wasused to terminate the reaction. Then optical density of each well was measuredby ELIASA at a wavelength of410nm.The detection of the cell cycle:1) The HDPCs of the4th passage that grewwell were chosen to prepare cells suspension of5×10⁴/ml and were culturedin25cm2plastic culture flask with DMEM containing15%of FBS anddivided into two groups: control group and experimental group, each group of3bottles. MTT assay was used to investigate the effect of10-8mol/LSimvastatin on proliferation of the cells of the experimental group at48h. Meanwhile, an equal amount of DMEM containing15%of FBS was addedinto the bottles of the control group.3) Trypsin was used to digest the cellsbefore they were collected into separate centrifugal tubes，fixed with75%iceethanol and kept in a refrigerator at4℃．4)100μl cell suspension was taken tobe incubated at room temperature of30min with400μl fluorescent dyecontaining1%of RNA enzymes，5%of Triton–100and5mg/L PI. Flowcytometry was used to dectec the cell cycle.The detection of the influence of Simvastatin on mineralized noduleformation of HDPCs by alizarin red staining: the HDPCs of the4th passagethat grew well were chosen to prepare cells suspension of2×10⁴/ml and theninoculated into a6-well plate. The HDPCs were divided into2groups, eachgroup of3wells: the group not induced and the10-7mmol/LSimvastatin-treating group induced by mineralized. The formulation of themineralized nodules was measured by Alizarin red staining after the HDPCswere cultured to20d.The single factor analysis of variance （One-way ANOVA） was made withSPSS13.0statistical software, significance test standard being a=0.05. Theindexes of the cell cycle were compared by independent sample t-test betweengroups.Results:1Immunohistochemical staining results showed that HDPCs were positivefor vimentin and negative for ke phosphatase （ALP） and the mineralization（p<0.05）of HDPCs was promoted after Simvastatin treatment.2Compared with control group, the proliferation of HDPCs （p <0.05） wasincreased after the low concentrations of Simvastatin treatment （10-9、10-8and10-7mol/L） but was inhibited after the high concentration ofSimvastatin treatment(10^-6mol/L). There was significant differencecomparing with the control group （p <0.05）.Conclusion:From above results we can draw the following conclusions: 1Simvastatin can promote the proliferation of HDPCs at low concentrationsand can inhibit their proliferation at high concentration;2Simvastatin can improve the alkaline phosphatase （ALP） activity and themineralization ability of HDPCs;3Simvastatin can promote DNA synthesis and mitosis of HDPCs.