Mouse incisors are capable of continuously growing due to the renewal of dental epithelium stem cells and mesenchymal stem cells residing at the proximal ends. The transcription factor Sox9 plays important roles in maintaining the stem cells of hair follicles, retinal progenitor cells and neural crest stem cells. Whether Sox9 is involved during mouse incisor development is not reported yet. In this study, we examined the expression pattern of Sox9 during mouse incisor development by in situ hybridization and immunohistochemistry. Sox9 mRNA and protein showed similar expression pattern from embryonic day (E) 13.5 to postnatal (PN) day 10. At E13.5 and E14.5, Sox9 was strongly expressed in the dental epithelium. At E16.5, Sox9 started to be detected in the mesenchymal cells within the dental pulp, especially the dental pulp cells that adjacent to the labial cervical loop. Similarly with E14.5, Sox9 was strongly detected in the labial cervical loop, including the basal epithelium, the stellate reticulum and the outer enamel epithelium from E16.5 to PN10. The mesenchyme adjacent to the labial cervical loop also showed strong signal of Sox9. The spatiotemporal expression of Sox9 suggested its possible involvement during mouse incisor development.

Odontoblasts are derived from dental papilla mesenchymal cells and have an important role in defense against bacterial infection, whereas autophagy can recycle long-lived proteins and damaged organelles to sustain cellular homeostasis. Thus, this study explores the role of autophagy in odontoblast differentiation with lipopolysaccharide (LPS) stimulation in vitro and the colocalization of p-NF-κB and LC3 in caries teeth. The odontoblasts differentiation was enhanced through LPS stimulation, and this outcome was reflected in the increased number of mineralized nodules and alkaline phosphatase (ALP) activity. The expression levels of the autophagy markers LC3, Atg5, Beclin1 and TFE3 increased time dependently, as well along with the amount of autophagosomes and autophagy fluxes. This result suggests that autophagy was enhanced in odontoblasts cultured with mineralized-induced media containing LPS. To confirm the role of autophagy in differentiated odontoblasts with LPS stimulation, chloroquine (CQ) or rapamycin were used to either block or enhance autophagy. The number of mineralized nodules decreased when autophagy was inhibited, but this number increased with rapamycin treatment. Phosphorylated nuclear factor-κB (NF-κB) expression was negatively related to autophagy and could inhibit odontoblast differentiation. Furthermore, p-NF-κB and LC3 colocalization could be detected in cells stimulated with LPS. The nucleus translocation of p-NF-κB in odontoblasts was enhanced when autophagy was inhibited by Atg5 small interfering RNA. In addition, the colocalization of p-NF-κB and LC3 in odontoblasts and sub-odontoblastic layers was observed in caries teeth with reactionary dentin. Therefore, our findings provide a novel insight into the role of autophagy in regulating odontoblast differentiation by suppressing NF-κB activation in inflammatory environments.

Vascularization is essential for organ and tissue development. Teeth develop through interactions between epithelium and mesenchyme. The developing capillaries in the enamel organ, the dental epithelial structure, occur simultaneously by mechanisms of vasculogenesis and angiogenesis at the onset of dentinogenesis. The vascular neoformation in the dental mesenchyme has been reported to start from the cap stage. However, the mechanisms of vascularization in the dental mesenchyme remain unknown. In the hope of understanding the mechanisms of the formation of dental mesenchymal vasculature, mouse lower molar germs from embryonic day (E) 13.5 to E16.5 were processed for immunostaining of CD31 and CD34, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and transmission electron microscopy (TEM). In addition, the role of apoptosis for the vascularization in dental mesenchyme was examined by in vitro culture of E14.0 lower molars in the presence of the apoptosis inhibitor (z-VAD-fmk) and a subsequent subrenal culture. Our results showed that CD31- and CD34-positive cells progressively entered the central part of the dental papilla from the peridental mesenchyme. For TEM, angioblasts, young capillaries with thick endothelium and endothelial cells containing vacuoles were observed in peripheral dental mesenchyme, suggesting vasculogenesis was taking place. The presence of lateral sprouting, cytoplasmic filopodia and transluminal bridges in the dental papilla suggested angiogenesis was also occurring. Inhibition of apoptosis delayed the angiogenic vascularization of the dental papilla. Therefore, these data demonstrated that molar mesenchyme is progressively vascularized by mechanisms of both vasculogenesis and angiogenesis and apoptosis partially contributes to the vascularization of the dental papilla.

Odontogenesis consists of a series of consecutive tooth morphogenesis stages, in which apoptosis is involved to eliminate the unnecessary cells. Autophagy, a lysosome or endosome-mediated self-degradation process, is indicated to participate in embryogenesis and tissue morphogenesis associated with apoptosis. This study hypothesized that autophagy may be involved and associated with apoptosis in odontogenesis. The transcripts of autophagy-related genes (Atg5, Atg7, and Atg12) were positively detected in tooth germs at embryonic day (E) 14.5 and postnatal day (P) 5.5 by quantitative real-time PCR. The protein expression of Atg5–Atg12 conjugate and lipidation of LC3 (microtubule-associated protein 1 light chain 3, autophagic marker) were revealed in the developing tooth germs by western blot. Meanwhile, LC3 was immunolocalized in the enamel organ and dental papilla at embryonic stages (E13.5–E18.5), especially stage E14.5 cervical loop and the PEK that facing the mesenchyme. At postnatal stages (P1.5–P15.5), besides the dental epithelium cells, LC3 was detected in the differentiating and differentiated odontoblasts, dental follicle cells, and Hertwig’s epithelium root sheath cells. Moreover, double-immunofluorescence analysis revealed the partial colocalization of LC3 and TUNEL signal in the E14.5 PEK that facing the mesenchyme, the E16.5 stratum intermedium and outer enamel epithelium, the P5.5 stratum intermedium and stellate reticulum. Nevertheless, LC3 was also found in non-apoptotic cells. Furthermore, the transmission electron microscopic images revealed the presence of autophagy, as well as the partial colocalization of autophagic vacuoles and apoptotic nuclei during tooth development. Our findings imply the developmental appearance of autophagy and its partial colocalization with apoptosis during odontogenesis.

Odontoblasts are terminally differentiated cells that play a vital role in dentinogenesis. The differentiation of odontoblasts is regulated by a variety of genetic and epigenetic mechanisms. Our previous microRNA microarray studies verified that miR-338-3p was up-regulated during odontoblast differentiation. The purpose of this study was to determine the function of miR-338-3p during odontoblast differentiation. The upregulation of miR-338-3p expression during odontoblast differentiation was validated by qRT-PCR. Odontoblast differentiation was enhanced after over-expression of miR-338-3p, while a loss of function approach using a miR-338-3p inhibitor impaired odontoblast differentiation. Bioinformatic analysis identified Runx2 as a potential target of miR-338-3p. Overexpression of miR-338-3p caused a decreased in the expression of Runx2 at both mRNA and protein levels, while Runx2 expression increased after treatment with miR-338-3p inhibitors. Furthermore, the activity of a luciferase reporter plasmid containing the 3′-UTR of Runx2 was significantly suppressed by ectopic expression of miR-338-3p. These results suggested that miR-338-3p promotes odontoblast differentiation through targeting Runx2.

Odontoblasts are a type of non-proliferating and terminally differentiated cells that play an important role in the pulpo–dentinal complex. Mouse dental papilla cells (mDPCs), which can differentiate into odontoblast-like cells in vitro, have a limited life span. We combined the traditional strategy of “Cre/LoxP-based reversible immortalization” with a tamoxifen-regulated Cre recombination system to generate a tamoxifen-mediated reversibly immortalized mouse dental papilla cell line, mDPCET. mDPCs were sequentially transduced with a floxed SV40 T antigen-TK (SV40Tag-TK) and an ERT2CreERT2-expressing plasmid. Clonal-isolated SV40Tag- and Cre-positive cells showed modified growth characteristics and a significantly extended life span. When mDPCET cells were treated with 4-hydroxytamoxifen, ERT2CreERT2 translocated from the cytoplasm to the nucleus and caused the excision of SV40Tag-TK, which led to the reversion of mDPCETs. After the immortalization was reversed, the cells underwent replicative senescence and transitioned into a more differentiated state. Tamoxifen-mediated reversible immortalization, therefore, allows for the expansion of primary mDPCs, leads to the production of odontoblast-like cells that retain most odontoblast-specific properties, and can represent a safe and ready-to-use method due to its simple manipulation.

Our previous study identified the appearance of autophagy in developing tooth germs, and suggested its possible association with apoptosis in odontogenesis. Beclin1 was recently indicated to play a central role in bridging autophagy and apoptosis, and occupied a key position in the process of development. This study hypothesized that Beclin1 may be involved, and act as the molecular basis of the connection between autophagy and apoptosis in odontogenesis. Immunohistochemical analysis showed the spatiotemporal expression pattern of Beclin1 in odontogenesis from embryonic (E) day 13.5 to postnatal (P) day 5.5. At E stages, Beclin1 was mainly immunolocalized in the cytoplasm of the cells in the enamel organ. Meanwhile, the nucleus localization of Beclin1 was detected in part of the stellate reticulum, outer and inner enamel epithelium, especially at E16.5 and E18.5. At P stages, Beclin1 was detected in the cytoplasm of the odontoblasts, besides the dental epithelium cells. Triple immunofluorescence analysis showed the partial colocalization of Beclin1, autophagic marker LC3, or activated caspase-3 in the E14.5 tooth germs, especially the Beclin1+LC3+Caspase-3+ cells in the PEK. Furthermore, western blot analysis revealed that the full-length (60 kDa) and/or cleaved (50, 37, and 35 kDa) Beclin1 in the developing tooth germs. Taken together, our findings indicate that Beclin1 is involved, and might be responsible for the crosstalk between autophagy and apoptosis in mouse odontogenesis.

To obtain the caries experience and, plaque accumulation severity and pit and fissure morphology in first permanent molars in 7–8 children in Wuhan, as a reasonable prediction of caries risk and preventive attention in the future, a convenient sample of five primary schools in the vicinity of the Wuhan University School and Hospital of Stomatology was drawn. Two calibrated examiners orally examined all present grade 2 children in the classroom, using standard caries plaque and tooth morphology criteria. Dental caries was scored at enamel (D2) and dentine (D3) for tooth and surface level. Independent variables were age, gender and school. Data analysis used analysis of variance and t-test. The sample comprised 1 043 7- and 8-year-olds. The prevalence of dental caries in permanent dentition was 8.7% and in primary dentition, 68.7%. Mean Decayed, Missing, Filled Teeth/S (DMFT/S) scores were 0.11 and 0.14, respectively. Mean dmft/s scores were 2.8 and 5.0. The d-component constituted 75% of the d3mft index, while enamel carious lesions constituted 36% of the total number of carious lesions (d2,3-component). Prevalence of medium and deep pits and fissures was 84.6%. Prevalence of medium and severe plaque accumulation was 67.4%. Prevalence of dental caries in the deciduous and permanent dentitions of 7- to 8-year-old children was high. Deep pits and fissures in high caries risk children should be sealed.

The root apex of the tooth elongates until the completion of root development. Although the signaling molecules inducing root elongation have been studied, the characteristic of the cells having the ability to maintain the root elongation remains unclear. This study aimed to investigate the characteristics of the cells involved in the root elongation. Octamer-binding factor 3/4 (Oct3/4) is known as one of the key regulators in maintaining the pluripotency and self-renewal properties of embryonic stem cells. Bmi1, the polycomb-group transcriptional repressor, has emerged as a key regulator in several cellular processes including stem cell self-renewal and cancer cell proliferation. At the beginning of root formation, ameloblasts expressed Oct3/4 in the nucleus, except in the apex of the cervical loop, in which Bmi1and cyclinD were expressed. At PN6, the expression of Oct3/4 in the ameloblasts shifted from the nucleus to the cytoplasm, whereas ameloblastin-negative Hertwig’s epithelial root sheath (HERS) cells expressed Bmi1 and cyclinD. By PN10, the cells in the apex of HERS began to express Oct3/4 in their nucleus, whereas Bmi1 and cyclinD began to decrease in their expressions. The odontoblasts consistently expressed Oct3/4 in their cytoplasm. Our results suggest that (1) Oct3/4 creates the border between the ameloblasts from the proliferative region of HERS, (2) Bmi1-positive cells would be one of the candidates resulting in root elongation and (3) the Oct3/4 expression in the cytoplasm of odontoblasts may be related to maintain the odontoblastic characteristics.

Dentin sialoprotein (DSP) is a major non-collagenous protein in dentin. Mutation studies in human, along with gene knockout and transgenic experiments in mice, have confirmed the critical role of DSP for dentin formation. Our previous study reported that DSP is processed into fragments in mouse odontoblast-like cells. In order to gain insights into the function of DSP fragments, we further evaluated the expression pattern of DSP in the mouse odontoblast-like cells using immunohistochemistry and western blot assay with antibodies against the NH2-terminal and COOH-terminal regions of DSP. Then, the distribution profiles of the DSP NH2-terminal and COOH-terminal fragments and osteopontin (OPN) were investigated in mouse teeth at different ages by immunohistochemistry. In the odontoblast-like cells, multiple low molecular weight DSP fragments were detected, suggesting that part of the DSP protein was processed in the odontoblast-like cells. In mouse first lower molars, immunoreactions for anti-DSP-NH2 antibody were intense in the predentin matrix but weak in mineralized dentin; in contrast, for anti-DSP-COOH antibody, strong immunoreactions were found in mineralized dentin, in particular dentinal tubules but weak in predentin. Therefore, DSP NH2-terminal and COOH-terminal fragments from odontoblasts were secreted to different parts of teeth, suggesting that they may play distinct roles in dentinogenesis. Meanwhile, both DSP antibodies showed weak staining in reactionary dentin (RD), whereas osteopontin (OPN) was clearly positive in RD. Therefore, DSP may be less crucial for RD formation than OPN.

The aim of this study is to investigate the influence of different posts on the fracture mechanics of endodontically-treated teeth with open apex. Forty-eight human maxillary anterior teeth were collected, and the root was transversely sectioned 12 mm under the cementoenamal junction (CEJ). These samples were then randomly divided into two groups, i.e., minor diameter open apex root (group A) and major diameter open apex root (group B), with mineral trioxide aggregate (MTA) placed into the apical 4 mm in the root canals. Subsequently, both groups were respectively further divided into three subgroups as follows: fiber-post (subgroup 1), metal post (subgroup 2) and non-post (subgroup 3) group. Teeth were restored with a composite resin crown and tested by using a universal testing machine at the rate of 1 mm/min cross-head. Values of the maximum fracture resistance and failure patterns were recorded and compared among all subgroups. In addition, the changes of MTA properties were carefully examined via X-ray photography. Our results indicate that (1) In group A, the mean value of fracture resistance for teeth restored with fiber posts were statistically higher than that with either metal post or non-post; (2) In group B, there was no statistically significant difference in the mean value of fracture resistance among three subgroups; (3) No statistical significance in the mean value of fracture resistance was found between group A and group B; (4) The failure modes of most samples (58%) were irreparable; (5) MTA in two teeth developed cracks after loading tests. In conclusion, endodontically-treated teeth restored with fiber posts are more resistant to fracture than those restored with either metal posts or non-post, and most of the fracture modes are catastrophic in nature.

The present study was designed to investigate the direct role of Shh molecule on cytodifferentiation and cusp formation. Affi-gel blue beads soaked in exogenous Shh-N, Shh antibody or BSA control protein were implanted between the epithelium and mesenchyme of isolated molar germs at the cap stage. The recombinants were grafted for culture under the kidney capsules respectively. In compared to the control, additional Shh-N protein could not enhance the ameloblasts and odontoblasts differentiation of the explanted tooth germs. While, application of Shh antibody retarded these events. After 4 weeks of subrenal culture, the teeth dissected from the explants treated with Shh-N were multicuspid. Most of the teeth harvested from the Shh antibody group were small and single irregularly shaped cusp was visible. The main cusp height in this group was reduced. The results indicated Shh signaling pathway is critical for odontoblast and ameloblast differentiation and patterns cusp formation.

Our previous studies have demonstrated that KLF4 is a critical transcription factor that promotes the odontoblastic differentiation of dental papilla cells. Klf4 mRNA was found to be regulated by multiple microRNAs (miRNAs). Competitive endogenous RNAs (ceRNAs) are a group of transcripts post-transcriptionally regulating each other by competing for their common miRNAs. However, the regulation of Klf4 by ceRNAs in odontoblastic differentiation remains unknown. In this study, we predicted a group of potential Klf4 ceRNAs with bioinformatics approach, and examined the expression of Klf4 and five interested potential ceRNAs including Sp1 using real-time PCR during odontoblastic differentiation of mDPC6T. The expression levels of both Sp1 and Klf4 were significantly upregulated during this process. In situ hybridization verified that Sp1 was co-expressed with Klf4 in the differentiating and the mature odontoblasts in vivo. Knockdown of Sp1 using siRNA resulted in a significant reduction of Klf4 and vice visa. This interaction was further confirmed to be miRNA dependent. Common miRNAs of Klf4 and Sp1 were predicted, among which miR-7a, miR-29b and miR-135a were able to downregulate both Klf4 and Sp1 expression after their separate overexpression in the mDPC6T cells. Dual luciferase assays showed that these miRNAs separately regulated the 3′UTRs of both Klf4 and Sp1, and the down-regulation of Klf4 3 ’UTR by Sp1 siRNA was abolished when these three miRNAs’ binding sites were mutated in the Klf4 3 ’UTR. Therefore, our results indicate that Sp1 functions as a ceRNA of Klf4 during odontoblastic differentiation through competing for miR-7a, miR-29b and miR-135a.