Melatonin-Medicated Neural JNK3 Up-Regulation Promotes Ameloblastic Mineralization.

Introduction: Melatonin, an endogenous neurohormone, modulates the biological circadian rhythms of vertebrates. It functions have been reported in previous stomatological studies as anti-inflammation, antioxidant, osseointegration of dental implants and stimulation to dental pulp stem cells differentiation, but its role in ameloblastic differentiation and mineralization has been rarely studied. Objective: To reveal the effects of melatonin on the mineralization of ameloblast lineage cells (ALCs), and to identify the change in gene expression and the potential mechanism based on ribonucleic acid sequencing (RNA-seq) analysis. Method: ALCs were induced in melatonin-conditioned medium. After 7-days culture, Western blot, real-time PCR, alkaline phosphatase (ALP) activity test, RNA-seq were accordingly used to detect the change in molecular level. After 1-month odontogenic induction in melatonin medium, Alizarin Red-S (ARS) staining showed the changes of mineral nodules. Differentially expressed genes (DEGs), enrichment of functions and signaling pathways analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) database were performed. The JNK3 antagonist (JNK3 inhibitor IX, SR3576) and ß-arrestin1 (Arrb1) overexpression were applied to confirm the fluctuation of melatonin-medicated JNK3 and Arrb1 expression. Results: In this study, we found out melatonin contributed to the ameloblastic mineralization, from which we can observed the elevated expression of enamel matrix protein, and increased ALP activity and mineralized nodules formation. RNA-seq analysis showed the up-regulation of neural JNK3 and down-regulation of Arrb1 in ALCs. Meanwhile, phosphorylated JNK3 deficiency (phosphorylated JNK3 inhibitor---SR3576 added to culture medium) led to mineralization delay, and Arrb1 overexpression proved Arrb1 takes bridge between melatonin receptors (MTNR) and JNK3 in MAPK signaling pathway.

hsa_circ_0026827 Promotes Osteoblast Differentiation of Human Dental Pulp Stem Cells Through the Beclin1 and RUNX1 Signaling Pathways by Sponging miR-188-3p.

Previous studies have found that circular RNA (circRNA) hsa_circ_0026827 plays a role during osteoblast differentiation, but the mechanism is unclear. The aim of this study was to illuminate the role of hsa_circ_0026827 in human dental pulp stem cells (DPSCs) during osteoblast differentiation. The results show that hsa_circ_0026827 expression significantly increased during osteoblast differentiation, while knockdown of hsa_circ_0026827 suppressed DPSC-derived osteoblast differentiation. microRNA (miRNA) expression profile analysis showed that downregulation of hsa_circ_0026827 promoted miR-188-3p expression. miR-188-3p downregulation restored osteogenic differentiation of DPSCs after hsa_circ_0026827 was silenced. Luciferase reporter assays verified that miR-188-3p was the target of hsa_circ_0026827 and also demonstrated that Beclin1 and RUNX1 were miR-188-3p downstream targets. miR-188-3p overexpression suppressed DPSC osteogenic differentiation by targeting Beclin-1-mediated autophagy and runt-related transcription factor 1 (RUNX1). In vivo studies using a heterotopic bone model also found that hsa_circ_0026827 overexpression plays an important role in promoting heterotopic bone formation. In conclusion, our research indicates that hsa_circ_0026827 promotes osteoblast differentiation of DPSCs via Beclin1 and the RUNX1 signaling pathways by sponging miR-188-3p, which suggests novel therapeutics for osteoporosis treatment.

Applicability of newly derived second and third molar maturity indices for indicating the legal age of 16 years in the Southern Chinese population.

In China, forensic age estimation in the living has become increasingly important in these few years. Due to increasing juvenile crimes, the age of 16 years has become the legal age threshold for determining whether the juvenile criminal law or adult criminal law applies to the citizen. This study aimed to assess new cut-off values of the third molar maturity index (I3M) and second molar maturity index (I2M) at the legal age threshold of 16 years in Southern Chinese population and then to compare the applicability of the cut-off values of I3M and I2M we set for discriminating whether a subject is ≥ 16 years. Orthopantomograms (OPGs) of 671 healthy Southern Chinese subjects (332 males and 339 females), aged between 10 and 20 years, were studied. Logistic regression analysis was performed with chronological age (below 16 years and over 16 years) as a dichotomous dependent variable and, I3M, I2M, and sex as predictive variables. The high p-value for sex (p = 0.861) reveals that this factor was not statistically significant in assessing the legal age of 16. The cut-off values of I3M < 0.38 and I2M < 0.03 were identified and used to distinguish between individuals who were or were not aged ≥ 16 years. Compared with a single predictor (I3M) alone, combining I3M and I2M can be more reliable for determining whether an individual is aged more than 16 years. We found that accuracy was 88.52% (95% confidence interval CI, 86.11-90.94%) and sensitivity and specificity were 77.96% (95% CI, 74.82-81.10%) and 97.28% (95% CI, 96.04-98.51%), respectively. The estimated Bayes post-test probability was 99.12% (95% CI, 98.41-99.83%). In conclusion, combining I3M and I2M may be useful in legal and forensic practices to determine the legal age of 16 years in Southern Chinese individuals.

The Circular RNA circRNA124534 Promotes Osteogenic Differentiation of Human Dental Pulp Stem Cells Through Modulation of the miR-496/ß-Catenin Pathway.

Circular RNAs (circRNAs) have been found to be a crucial role in stem cell-associated bone regeneration. However, the functions and underlying mechanisms of circRNAs in the osteogenic differentiation of human dental pulp stem cells (hDPSCs) remain largely unclear. We found that overexpression of circRNA124534 unexpectedly promoted DPSCs osteogenesis in vitro and in vivo. Our results confirmed circRNA124534, acting as a miRNA sponge, directly interacts with miR-496 and consequently regulates ß-catenin, which in turn exerts osteogenesis of DPSCs. Enforced expression of miR-496 reversed the osteogenesis of circRNA124534, and suppression of miR-496 enhanced the osteogenic differentiation of DPSCs by promoting ß-catenin. In conclusion, our findings demonstrate functions of circRNA124534 in modulating osteogenic differentiation through the miR-496/ß-catenin pathway; thus, providing a novel potential target for therapy.