Study On NF-κB Pathway Mechanism During The Differentiation Of Human Stem Cells From Apical Papilla Induced By Mineral Trioxide Aggregate

Mineral trioxide aggregate (MTA), as a bioactive material, has been widely used inthe clinical apexification. To date, the effects MTA on the proliferation anddifferentiation of human stem cells from apical papilla (SCAPs), and the potentialmechanisms remain unclear. This paper was designed to answer these questions fromthree different aspects.Part Ⅰ. Concentration determination of MTA conditionedmedium and its effects on the proliferation of SCAPsObjective To select the optimal concentration of MTA conditioned medium andinvestigate the effects on the proliferation of SCAPs.Methods SCAPs was cultured in α-MEM medium supplemented with differentconcentrations of MTA conditioned medium (MTA-CM) in vitro. And the optimalconcentration of MTA-CM was determined by alkaline phosphatase (ALP) activityassay. The alizarin red staining and CPC assay were used to confirm the optimalconcentration. Methyltetrazolium (MTT) assay and flow cytometry (FCM) analysiswere performed to evaluate the proliferation activity of MTA-induced SCAPs. Results ALP activity assay revealed that MTA at2mg/mL was the optimalconcentration during the differentiation of MTA-treated SCAPs. When treated with2mg/mL MTA, mineralization ability in SCAPs was also significantly upregulated. Ascompared with control group, the proliferation activity of SCAPs was not affected byMTA at2mg/mL.Conclusions2mg/mL MTA is the optimal concentration in triggering theodonto/osteogenic differentiation of SCAPs.2mg/mL MTA conditioned mediumcan promote the mineralization ability of SCAPs without affecting its proliferationcapacity in vitro.Part Ⅱ. Effects of MTA on the differentiation andmorphogenesis of SCAPsObjective To observe the effects of MTA on odontogenic/osteogenic differentiationand morphogenesis of SCAPs.Methods Odonto/osteoblastic genes and proteins (ALP/ALP, dentinsialophosphoprotein(DSPP)/dentin sialoprotein(DSP), runt-related transcriptionfactor2(RUNX2)/RUNX2and osteocalcin(OCN)/OCN) were assessed by real-timeRT-PCR and Western blot respectively. Moreover, SCAPs pellets with or withoutMTA treatment was carefully recombined with the scaffold of root canal andtransplanted into the renal capsules of adult rat hosts to evaluate their morphogeniccapacity in vivo.Results The protein/gene expressions of OCN, RUNX2, and DSP in MTA-treatedgroup were significantly higher than those in control group in vitro. In addition, MTA-treated SCAPs pellets in vivo can form the regular dentin-pulp-like complex,while untreated SCAPs pellets can not.Conclusions MTA can promote the differentiation of SCAPs intoodontogenic/osteogenic lineages in vitro, and facilitate the dentinogenesis of SCAPsin vivo.Part Ⅲ. NF-κB pathway mechanism embedding in thedifferentiation of MTA-treated SCAPsObjective To explore the NF-κB pathway mechanism during the differentiation ofSCAPs induced by MTA.Methods Western blot was used to detect the status of NF-κB pathway according tothe expression of several key proteins (NF-κB P65, IκB-α, phosphorylation NF-ΚBP65and phosphorylation IκB-α). And immunofluorescence staining was used toobserve the nuclear translocation of NF-κB P65.Results P65was transferred from cytoplasm to nuclei, as indicated by Western blotand immrnofluorescencestaining. p-P65level was increased at0.25h and keptclimbing at0.5h, and then gradually droped. p-IκB-α was rapidly risen at0.25h andthen decreased. IκB-α was slightly decreased at first and regained later. Thesefindings suggest that the NF-κB pathway was activated during the treatment ofMTA.Conclusions NF-κB pathway was activated instantly when SCAPs were induced byMTA, indicating that MTA-mediated odonto/osteoblastic differentiation of SCAPsmay be connected to the activation of NF-κB pathway.