Role And Mechanisms Of BMP2 In The Odontoblastic Differentiation Of The Stem Cells From The Apical Papilla

Stem cells from the apical papilla (SCAPs) have multi-lineage differentiation potential and can be differentiated into odontoblast. SCAPs play an important role in the root dentin development of immaturate permanent teeth. Human bone morphogenetic protein 2 (BMP2) has been proved to participate in the regulating odontogenic differentiation of mesenchymal stem cells during the process of tooth development. However, little is know about the role and related mechanisms of BMP2 on the proliferation and differentiation of SCAPs. This study aimed to evaluate the role and mechanisms of BMP2 in the odontoblastic differentiation of the stem cells from the apical papillia in vitro.Main methods and results1. Isolation, expandation and identification of SCAPs.SCAPs were isolated and cultured from immature third molar apical papilla. We used immunofluorescence staining of STRO-1, flow cytometric (FCM) analysis (STRO-1, CD24, CD45, CD146), and mineralized and adipogenic induction to identify the SCAP characteristics. Immunocytochemical staining and FCM analysis showed that the cells derived from apical papilla contained mesenchymal stem cell populations. Besides, the isolated cells can be induced to osteo/odontogenic and adipogenic differentiation under defined culture conditions.2. The effects of BMP2 on proliferation and differentiation of SCAPs.The effect of BMP2 on proliferation of SCAPs was analyzed by a cell counting kit-8 (CCK-8). The ALP activity was measured on the 3rd and 5th days using an ALP kit. The mRNA and protein of dentin sialophosphoprotein (DSPP), runt-related transcription factor 2 (RUNX2), bone sialoprotein (BSP), and osteocalcin (OCN) were evaluated by Real-time quantitative polymerase chain reaction (Real-time qPCR) and western blotting. Results showed that BMP2 at different concentration did not exhibit a prominent effect on SCAP proliferation (p<0.05). The ALP activity was enhanced in the BMP2 groups than in the control group on both days 3 and 5 and the 200 ng/ml and 400 ng/ml BMP2 group exhibited the most significant effect. There is no statistical difference between the two groups, so we chose the 200ng/ml as the experimental concentration. Real-time PCR assays and western blotting showed that mRNA and proteins expression of osteo/odontogenic genes, such as DSPP, Runx2, BSP, and OCN, in the BMP2 groups were up regulated compared with the control group on day 5. The results demonstrated that BMP2 promoted the odontoblast differentiation of SCAPs.3. The effect of BMP2 on MAPK signaling pathway and the related mechanisms of BMP2 on odontoblast differentiation of SCAPs.For the signaling pathway analysis, p38, ERK and JNK kinase activation was examined using western blotting. Phosphorylation of ERK, p38 and JNK were increased. For the mechanisms study, cells were pretreated with a signaling pathway inhibitor (SB203580, an inhibitor of p38, U0126, an inhibitor of ERK and SP600125, an inhibitor of JNK) and then cultured with BMP2.. After pretreatment with inhibitors, ALP, osteo/odontogenic mRNA and proteins (RUNX2, DSPP, OCN, and BSP) expression decreased. In conclusion, the MAPK signaling pathways play an important role in BMP2-induced odonto/osteoblastic SCAP differentiation.