The Effects Of Calcium Hydroxide On The Proliferation Of Stem Cells From Apical Papilla Of Human

Objective:Stem cells from apical papilla（SCAP）was important seed cells in root formation and tooth regeneration.The cells were successfully isolated and cultivated from apical papilla of young human immature teeth by enzyme digestion method organized piece of adherent explanted tissue culture in vitro,then explored the characteristics and detected the effects of different concentration of calcium hydroxide on the proliferation of them,so as to provide the theoretical basis of calcium hydroxide in clinical use of root development and regeneration.Methods:Premolars and third molars were collected from young healthy orthodontic patients,then separated the apex tissue block of dental papilla.Use enzyme digestion method organized piece of adherent explanted tissue culture to isolate primary stem cells,then purified cells with limited dilution monoclonal method,and use flow cytometry instrument to detect expression of the mesenchymal stem cell surface markers STRO-1,CD146,CD90,CD45,and use crystal violet staining method to detect the colony forming unit-fibroblastic.Use MTT method to determine the OD₄₉₀90 value of cells at 1,3,5,7,9 day and draw the growth curve of cells.After 2w and 4w culture respectively in vitro,use Oil Red-O and Alizarin Red staining to confirm adipogenic differentiation and osteogenic differentiation of SCAP.Take growth in good condition of SCAP to 96-well plate,after 24h hunger cultivation,replaceα-MEM medium which contain 0.1%,0.5%,1%,5%,10%,15%calcium hydroxide.Then divided the wells into two groups randomly,each group has six duplicated holes,detected OD₄₉₀ value at 1,3,5,7,9 day for 3 times by MTT method.Results:The cells were observed swum out of tissue block edge under inverted microscope after 3d,and grow around the tissue block.The morphology of cells were relatively uniform as small volume and short spindly,a few of them were stellated fibroblast-like.Purified cells extend the typical morphology of fibroblast cells as short spindle,small volume,and shown active proliferative capacity.The growth curve of SCAP had a S shape,and colony-forming unit was 20.96%.Mesenchymal stem cell markers like STRO-1,CD146,CD90 were expressed positively,the ratio were15.69%,54.06%,99.93%respectively,while CD45 was expressed negatively.After osteogenic differentiation in vitro by 2 weeks and 4 weeks,ALP staining was positively and mineralized nodules were detected by Alizarin Red staining.After adipogenic differentiation in vitro by 3 circulations,red lipid droplets were detected.Compared with control group at 1d and 3d,the cell density of each group was no obvious different（P>0.05）;At 5d,the cell density of concentration of calcium hydroxide below 5%was no obvious different（P>0.05）,while there was obvious different（P<0.05）of high concentration above 10%;To 7d and 9d,the cells density of each group was statistically significant（P<0.05）;Conclusition:1.This experiment successfully cultured stem cells from the apical papilla of human,detected the expression of mesenchymal stem cell surface markers and the multi-lineage differentiation in vitro.Confirmed the SCAP of human has stem cells phenotype characteristics and multi-directional differentiation potential,and could be used as tissues engineering seed cells,the cell sources of dental pulp regeneration.2.The inhibit ability of calcium hydroxide to SCAP proliferation was higher as extending of time and concentration.