RESUMEN
BACKGROUND: Stem cells from apical papilla (SCAP) are a subpopulation of mesenchymal stem cells (MSCs) isolated from the apical papilla of the developing tooth root apex of human teeth. Because of their osteogenic/dentinogenic capacity, SCAP are considered as a source for bone and dentin regeneration. However, little is understood about the molecular mechanism of osteogenic/dentinogenic differentiation of SCAP. Phosphoinositide 3 kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) signal pathway participates in regulating the differentiation of various cell types, such as MSCs. In this study, we examined the role of the PI3K-AKT-mTOR signal pathway in the osteogenic/dentinogenic differentiation of SCAP. Moreover, we challenge to fabricate scaffold-free SCAP-based spheroidal calcified constructs. METHODS: SCAP were pretreated with or without small interfering RNA for AKT (AKT siRNA), PI3K inhibitor LY294402, and mTOR inhibitor rapamycin and were cultured under osteogenic/dentinogenic differentiation to examine in vitro and in vivo calcified tissue formation. Moreover, SCAP-based cell aggregates were pretreated with or without LY294402 and rapamycin. The cell aggregates were cultured under osteogenic/dentinogenic condition and were analyzed the calcification of the aggregates. RESULTS: Pretreatment with AKT siRNA, LY294402, and rapamycin enhances the in vitro and in vivo calcified tissue-forming capacity of SCAP. SCAP were fabricated as scaffold-free spheroids and were induced into forming calcified 3D constructs. The calcified density of the spheroidal constructs was enhanced when the spheroids were pretreated with LY294402 and rapamycin. CONCLUSIONS: Our findings indicate that the suppression of PI3K-AKT-mTOR signal pathway plays a role in not only enhancing the in vivo and in vitro osteogenic/dentinogenic differentiation of SCAP, but also promoting the calcification of scaffold-free SCAP-based calcified constructs. These findings suggest that a suppressive regulation of PI3K-AKT-mTOR signal pathway is a novel approach for SCAP-based bone and dentin regeneration.
Asunto(s)
Papila Dental/citología , Dentinogénesis , Células Madre Mesenquimatosas/citología , Osteogénesis , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Regeneración Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Dentina/metabolismo , Dentinogénesis/efectos de los fármacos , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Sirolimus/farmacología , Esferoides Celulares/citología , Esferoides Celulares/efectos de los fármacos , Regulación hacia Arriba , Adulto JovenRESUMEN
Nitric oxide (NO) is thought to play a pivotal regulatory role in dental pulp tissues under both physiological and pathological conditions. However, little is known about the NO functions in dental pulp stem cells (DPSCs). We examined the direct actions of a spontaneous NO gas-releasing donor, NOC-18, on the odontogenic capacity of rat DPSCs (rDPSCs). In the presence of NOC-18, rDPSCs were transformed into odontoblast-like cells with long cytoplasmic processes and a polarized nucleus. NOC-18 treatment increased alkaline phosphatase activity and enhanced dentin-like mineralized tissue formation and the expression levels of several odontoblast-specific genes, such as runt related factor 2, dentin matrix protein 1 and dentin sialophosphoprotein, in rDPSCs. In contrast, carboxy-PTIO, a NO scavenger, completely suppressed the odontogenic capacity of rDPSCs. This NO-promoted odontogenic differentiation was activated by tumor necrosis factor-NF-κB axis in rDPSCs. Further in vivo study demonstrated that NOC-18-application in a tooth cavity accelerated tertiary dentin formation, which was associated with early nitrotyrosine expression in the dental pulp tissues beneath the cavity. Taken together, the present findings indicate that exogenous NO directly induces the odontogenic capacity of rDPSCs, suggesting that NO donors might offer a novel host DPSC-targeting alternative to current pulp capping agents in endodontics.