Isolation,cultivation And Identification Of Stem Cell From Apical Papilla Of Canine

Objective In vitro successfully isolate and cultivate stem cell from apical papilla(SCAP) from apical papilla of canine immature teeth by explanted tissue culture, exploring the self-renewal and multi-lineage differentiation ability,to lay the foundation for the further research and application of SCAP.Methods Healthy immature teeth from canine were extracted to get apical papilla, isolating and cultivating primary cells by explanted tissue culture. Cell morphology and growth characteristics were detailedly observed and recorded.Cell surface markers were identified by flow cytometry.Colony-forming unit was counted by plate clone formation assay.Plasmosin-expressing was detected by cell immunofluorescence method.Their osteogenic and adipogenic potential were explored.Results Explanted tissue culture could steadily isolate and cultivate adherent cells from apical papilla of canine.They were clonogenic cells,short spindly or stellated fibroblast-like cells,whose colony-forming unit was counted(116.83+12.608)every 600 cells.Mesenchymal stem cell markers like STRO-1,CD90,CD24 were positively expressed,while CD34 was negatively expressed.Immunofluorescence analysis showed cells positive expression of Nestin and Vimentin, negative or very weakly positive expression of dentin sialoprotein(DSP). After osteogenic differentiation in vitro, mineralized nodules were detected by Alizarin Red staining and ALP and DSP were positively expressed.After adipogenic differentiation,lots of red lipid droplets were detected.After induction,differentiation markers, including BSP,DSP,RUNX2,LPL,were positively expressed by reverse transcriptionpolymerase chain reaction(RT-PCR).Conclusition Explanted tissue culture could steadily isolate and cultivate SCAP from apical papilla of canine,which has the ability of high proliferation and multi-lineage differentiation.The experiment is a preliminary study on the animal in vitro,further guiding research on application of SCAP and laying the foundation for the effective application of SCAP in pulp tissue engineering.