Triiodothyronine Promotes Osteogenesis Of Stem Cells From Apical Papilla Via ERK/MAPK Signal Pathway

PartⅠ.Isolation,culture and identification of stem cell from apical papilla（SCAPs）as well as selecting optimal concentration of T3Objective:The aim of this experiment is to isolate,culture and identify stem cell from apical papilla and select the optimal stimulation concentration of T3.Methods:The apical non-diagnosed permanent molars were collected.Then we combined with the enzyme digestion method and tissue block attachment method to culture the primary SCAPs and pass them to p3 to p5 for the flowing experiments.Induced multi-directional differentiation experiments were used to verify its differentiation potential.FCM was used to detect the surface molecular markers.The stock solution of 3,5,3’-Triiodothyronine（T3）was dissolved in 1.0 mol/L Na OH and used to prepared T3 conditioned medium with different concentration of T3.Then,the alkaline phosphatase activity of each concentration group was detected to choose optimal concentration to used in the following experiments.In addition,CCK-8 kit was used to draw the proliferation curve,and the cell cycle was detected by flow cytometry.Results:In this experiment,spindle cells were isolated from the papilla tissue of immature permanent molar root apex.The cells can be induced into osteogenic,adipogenic and chondrogenic differentiation.FCM showed that cells were highly expressed CD29 and CD90,but were lowly expressed CD34 and CD45.After SCAPs were stimulated with T3 conditioned medium with multiple concentrations for 5 d,the expression and activity of ALP were measured.The results showed that 10^-9mol/L T3 group shown higher ALP activity than control group and any other T3groups at different concentrations.The result of CCK-8 and FCM showed that 10^-9mol/L T3 had no significant effect on the proliferation ability of SCAPs.Conclusions:The cells isolated from papilla tissue were stem cell from apical papilla.10^-9mol/L T3 may promote osteogenic differentiation of SCAPa and does not significantly affect the proliferation of SCAPs.PartⅡ.Effects of T3 on osteogenic differentiation of SCAPsObjective:To explore the effects of T3 on the osteogenic differentiation of SCAPs.Methods:After stimulation with T3 conditioned medium,the expression level of osteogenic differentiation-related genes was detected by real-time RT-PCR,and the expression of osteogenic differentiation-related proteins was detected by Western blot and immunofluorescence staining.ALP staining and alizarin red staining were used to verify the effect of T3 on the mineralization ability of SCAPsResults:Real-time RT-PCR showed that the expression level of osteogenic differentiation-related genes（RUNX2,ALP,DMP1,OSX,OCN）was significantly increased compared with the control group after induction of T3 conditioned medium.The results of Western blot and immunofluorescence staining showed that the expression levels of osteogenic differentiation-related proteins（RUNX2,ALP,DMP1,OSX,OCN）increased after induction of T3 conditioned medium.ALP staining showed that deep-stained cells increased significantly after induction with T3conditioned medium,while in alizarin red staining,the T3 treated group had more mineralized nodules than the control group.Conclusions:10^-9mol/L T3 promotes osteogenic differentiation of SCAPs.Part Ⅲ.The effect of T3 on the MAPK signaling pathwayObjective: To explore the role of MAPK signaling pathway on T3-mediated osteogenic differentiation of SCAPs.Methods: After T3 treated,Western blot was used to detect the changes in phosphorylation levels of ERK,JNK and p38,and the effects of corresponding pathway inhibitors on T3-induced SCAPs osteogenic differentiation were examined.Results: After T3 conditioned medium induction,the level of p-ERK increased at 15 min.However,The phosphorylation level of P38 and JNK had no obvious change.After U0126 was used to inhibit the ERK pathway,the expression of osteogenic differentiation-related proteins decreased compared to the T3 induced group.The number of deep stained cells ALP staining and calcium nodules in alizarin red staining was reduced compared to the T3 treated group.Conclusions: The ERK signaling pathway plays a regulatory role in T3-mediated osteogenic differentiation of SCAPs.