Dental Papilla Cells Research Articles

A WWP2–PTEN–KLF5 signaling axis regulates odontoblast differentiation and dentinogenesis in mice

WW domain–containing E3 Ubiquitin-protein ligase 2 (WWP2) has been found to positively regulate odontoblastic differentiation by monoubiquitinating the transcription factor Kruppel-like factor 5 (KLF5) in a cell culture system. However, the in vivo role of WWP2 in mouse teeth remains unknown. To explore this, here we generated Wwp2 knockout (Wwp2 KO) mice. We found that molars in Wwp2 KO mice exhibited thinner dentin, widened predentin, and reduced numbers of dentinal tubules. In addition, expression of the odontoblast differentiation markers Dspp and Dmp1 was decreased in the odontoblast layers of Wwp2 KO mice. These findings demonstrate that WWP2 may facilitate odontoblast differentiation and dentinogenesis. Furthermore, we show for the first time that phosphatase and tensin homolog (PTEN), a tumor suppressor, is expressed in dental papilla cells and odontoblasts of mouse molars and acts as a negative regulator of odontoblastic differentiation. Further investigation indicated that PTEN is targeted by WWP2 for degradation during odontoblastic differentiation. We demonstrate PTEN physically interacts with and inhibits the transcriptional activity of KLF5 on Dspp and Dmp1. Finally, we found WWP2 was able to suppress the interaction between PTEN and KLF5, which diminished the inhibition effect of PTEN on KLF5. Taken together, this study confirms the essential role of WWP2 and the WWP2–PTEN–KLF5 signaling axis in odontoblast differentiation and dentinogenesis in vivo.

Role of the Demethylase AlkB Homolog H5 in the Promotion of Dentinogenesis.

Dentinogenesis is a key process in tooth formation and is regulated by a series of pre- and post-transcriptional regulations. N6-methyl-adenosine (m6A), which is the most prevalent internal chemical modification that can be removed by the RNA demethylase AlkB homolog H5 (ALKBH5), has recently been reported to be involved in several biological processes. However, the exact function of ALKBH5-mediated m6A modification in tooth development remains unclear. Here, we showed that Alkbh5 was expressed in pre-odontoblasts, polarizing odontoblasts, and secretory odontoblasts. Alkbh5 overexpression in the mouse dental papilla cell line mDPC6T promoted odontoblastic differentiation. Conditional knockout of Alkbh5 in Dmp1-expressing odontoblasts led to a decrease in number of odontoblasts and increased pre-dentin formation. Mechanistically, RNA sequencing and m6A sequencing of Alkbh5-overexpressing mDPC6T cells revealed that Alkbh5 promoted odontoblast differentiation by prolonging the half-life of Runx2 transcripts in an m6A-dependent manner and by activating the phosphatidylinositol 3-kinase/protein kinase B pathway. Notably, the loss of Alkbh5 expression in odontoblasts impaired tertiary dentin formation in vivo. These results suggested that the RNA demethylase ALKBH5 plays a role in dentinogenesis.

3D-bioprinted Recombination Structure of Hertwig's Epithelial Root Sheath Cells and Dental Papilla Cells for Alveolar Bone Regeneration.

Three-dimensional (3D) bioprinting is an emerging method for tissue regeneration. However, promoting the epithelial-mesenchymal interaction (EMI), while maintaining the characteristics of epithelial cells has always been a challenge in tissue engineering. Since EMI acts as a critical factor in bone regeneration, this study aims to promote EMI by recombining epithelial and mesenchymal cells through 3D bioprinting. Hertwig’s epithelial root sheath (HERS) is a transient structure appeared in the process of tooth root formation. Its epithelial characteristics are easy to attenuate under appropriate culture environment. We recombined HERS cells and dental papilla cells (DPCs) through 3D bioprinting to simulate the micro-environment of cell-cell interaction in vivo. HERS cells and DPCs were mixed with gelatin methacrylate (GelMA) separately to prepare bio-inks for bioprinting. The cells/GelMA constructs were transplanted into the alveolar socket of Sprague-Dawley rats and then observed for 8 weeks. Hematoxylin and eosin staining, Masson staining, and immunohistochemical analysis showed that dimensional cultural pattern provided ideal environment for HERS cells and DPCs to generate mineralization texture and promote alveolar bone regeneration through their interactions. 3D bioprinting technology provides a new way for the co-culture of HERS cells and DPCs and this study is inspiring for future research on EMI model.

The glycoprotein Wnt6 regulates human dental papilla cells differentiation by canonical Wnt signaling pathway

ObjectiveThe aim of this study was to test the hypothesis in vitro and in vivo, that the glycoprotein Wnt6 can regulate human dental papilla cell differentiation by β-catenin signaling. DesignThe expression of Wnt6 was detected by quantitative polymerase chain reaction (qPCR). Wnt6 stealth RNA was used to knockdown the expression of Wnt6. The Wnt canonical signaling was detected by immunofluorescence staining, qPCR, and TOPflash/FOPflash dual-luciferase reporter assay. The differentiation was investigated by alkaline phosphatase staining or Alizarin Red staining after osteo/odontogenic medium culture and by Masson trichrome staining after subcutaneous transplantation. There are at least three samples in one group for each experiment. ResultsWnt6 protein and mRNA were high expressed in dental mesenchyme tissue and cells. In human dental papilla cells, Wnt6 over-expression could activate β-catenin dependent pathway, including β-catenin accumulation in cell nuclei, lymphoid enhancer factor 1 mRNA up-regulation, and enhanced β-catenin transcriptional activity. Wnt6 activated β-catenin pathway in a similar way to Wnt3a but at a lower level. Wnt6 inhibited human dental papilla cells differentiation as alkaline phosphatase activity in vitro, and promoted differentiation as mineralization after subcutaneous transplantation in vivo, as same trend as Wnt3a but at a lower level. The Wnt/β-catenin inhibitor XAV939 treatment attenuated Wnt6- or Wnt3a-induced human dental papilla cells mineralization. ConclusionsWnt6 activated β-catenin dependent pathway and regulated human dental papilla cells differentiation. Potential mechanism of Wnt6-regulated cell differentiation is the activation of Wnt/β-catenin signaling pathway.

The effect of melatonin on the mouse ameloblast-lineage cell line ALCs

Melatonin plays a critical role in promoting the proliferation of osteoblasts and the growth and development of dental papilla cells. However, the effect and mechanism of melatonin on the growth and development of ALCs still need to be explored. CCK8 assay was used for the evaluation of cell numbers. qRT-PCR was used to identify the differentially expressed genes in ALCs after melatonin treatment. The number and morphology of ALCs were investigated by confocal microscopy. Alkaline phosphatase assay and Alizarin red S staining were used for measuring mineralization. Then, we focused on observing the crucial factors of the signaling pathway by RNA-seq and qRT-PCR. Melatonin limited the cell number of ALCs in a dose-dependent manner and promoted the production of actin fibers. A high concentration of melatonin significantly promoted the mRNA levels of enamel matrix proteins and the formation of mineralized nodules. RNA-seq data showed that Wnt signaling pathway may be involved in the differentiation of ALCs under the influence of melatonin. This study suggests that melatonin plays a regulatory role in the cell number, differentiation, and mineralization of the ALCs, and then shows the relationship between the Wnt signaling pathway with the ALCs under melatonin.

KLF6 facilitates differentiation of odontoblasts through modulating the expression of P21 in vitro

Multiple signaling pathways are involved in the regulation of cell proliferation and differentiation in odontogenesis and dental tissue renewal, but the details of these mechanisms remain unknown. Here, we investigated the expression patterns of a transcription factor, Krüppel-like factor 6 (KLF6), during the development of murine tooth germ and its function in odontoblastic differentiation. KLF6 was almost ubiquitously expressed in odontoblasts at various stages, and it was co-expressed with P21 (to varying degrees) in mouse dental germ. To determine the function of Klf6, overexpression and knockdown experiments were performed in a mouse dental papilla cell line (iMDP-3). Klf6 functioned as a promoter of odontoblastic differentiation and inhibited the proliferation and cell cycle progression of iMDP-3 through p21 upregulation. Dual-luciferase reporter assay and chromatin immunoprecipitation showed that Klf6 directly activates p21 transcription. Additionally, the in vivo study showed that KLF6 and P21 were also co-expressed in odontoblasts around the reparative dentin. In conclusion, Klf6 regulates the transcriptional activity of p21, thus promoting the cell proliferation to odontoblastic differentiation transition in vitro. This study provides a theoretical basis for odontoblast differentiation and the formation of reparative dentine regeneration.

IGFs in Dentin Formation and Regeneration: Progress and Remaining Challenges.

Tertiary dentin results from the interplay between the host defense and dental injury or infection. Modern endodontics aiming vital pulp treatment take the tertiary dentin formation as the interim step, with the final goal of a physiological pulp-dentin like tissue regeneration. Dental pulp stem cells have been nominated for contributing to differentiating into odontoblast-like cells who are responsible for reparative dentin formation. Understanding the original dentin formation mechanism provides us a blueprint while exploring the reparative dentin formation mechanism builds bridge to bonafide pulp-dentin tissue regeneration. Among all the regulators, growth factors have long been revealed under the spotlight. The insulin-like growth factor (IGF) family has been implicated in critical events of inducing dentin formation, which is essential for pulp treatment. The expression of IGF family members including IGF1, IGF1R, IGF2, and IGF2R has been well characterized in dental papilla cells, dental pulp stem cells, and periodontal ligament cells. Recent studies indicated IGF binding to the receptors activated pathways, including MAPK pathway, and AKT pathway, orchestrated proliferation, and differentiation, and finally, contributed to dentin formation. This review summarizes the role of IGF family in dentin formation during tooth development and tertiary dentin formation during dentin-pulp repair and sheds light on key parts of research for future treatment improvements.

Dlx3 Ubiquitination by Nuclear Mdm2 Is Essential for Dentinogenesis in Mice

Dentin is a major mineralized component of teeth. Odontoblasts are responsible for synthesis and secretion of dentin matrix. Previously, it has been demonstrated in a cell culture system that the E3 ubiquitin ligase, murine double minute 2 (Mdm2), promotes odontoblast-like differentiation of mouse dental papilla cells (mDPCs) by ubiquitinating p53 and the odontoblast-specific substrate Dlx3. However, whether Mdm2 plays an essential role in vivo in odontoblast differentiation and dentin formation remains unknown. In this study, we investigated the in vivo functions of Mdm2 using Dmp1-Cre;Mdm2 flox/flox mice combined with multiple histological and molecular biological methods. The results showed that Mdm2 deletion in the odontoblast layer led to defects in odontoblast differentiation and dentin formation. Unexpectedly, specific inhibition of the Mdm2-p53 axis in wild-type mice by injection of a small-molecule inhibitor Nutlin-3a indicated that the role of Mdm2 in dentinogenesis was p53 independent, which was inconsistent with the previous in vitro study. In situ proximity ligation assay (PLA) showed that Mdm2 interacted with and ubiquitinated Dlx3 in the odontoblast nucleus of mouse molars. Dlx3 promoted the translocation of Mdm2 to the nucleus, and in turn, the nuclear Mdm2 mediated ubiquitination of Dlx3 and promoted the odontoblast-like differentiation of mDPCs. Dlx3 interacted with Mdm2 through its C-terminal domain. Deletion of the C-terminal domain of Dlx3 reversed the enhanced odontoblast-like differentiation and the activation of Dspp promoter mediated by overexpression of wild-type or nuclear Mdm2. Our findings suggest that nuclear Mdm2 mediates ubiquitination of the transcription factor Dlx3, which is essential for Dlx3 transcriptional activity on Dspp as well as subsequent odontoblast differentiation and dentin formation.

Mesenchymal Mycn participates in odontoblastic lineage commitment by regulating Kr\xfcppel-like Factor 4 (Klf4) in mice

BackgroundCommitment of mouse dental papilla cells (mDPCs) to the odontoblast lineage is critical for dentin formation, and this biological process is regulated by a complex transcription factor network. The transcription factor Mycn is a proto-oncogene that plays an important role in tumorigenesis and normal embryonic development. An early study revealed that Mycn is exclusively expressed in dental mesenchymal cells at E15.5, which implies a potential role of Mycn in dentinogenesis. However, the role of Mycn in dentin formation remains elusive. Thus, it is of considerable interest to elucidate the role of Mycn in dentin formation.MethodsMycnfl/fl; Osr2IresCre (MycnOsr2) and Mycnfl/fl; K14Cre (MycnK14) transgenic mice were generated, and micro-CT scans were performed to quantitatively analyse the volumetric differences in the molars and incisors of the mutants and their littermates. Mycn was also knocked down in vitro, and alkaline phosphatase (ALP) and alizarin red staining (ARS) were conducted. Cleavage under targets and tagmentation (CUT&Tag) analysis and dual luciferase assays were performed to identify direct downstream targets of Mycn. Immunofluorescence and immunochemistry staining and western blotting (WB) were performed to analyse the expression levels of potential targets. Quantitative PCR, WB, ALP and ARS were performed to test the rescue efficiency.ResultsMesenchymal ablation of Mycn (MycnOsr2) led to defective dentin formation, while epithelial deletion (MycnK14) had no obvious effects on tooth development. ALP and ARS staining revealed that the commitment capacity of mDPCs to the odontoblast lineage was compromised in MycnOsr2 mice. CUT&Tag analysis identified Klf4 as a potential direct target of Mycn, and a dual luciferase reporter assay verified that Mycn could bind to the promotor region of Klf4 and directly activate its transcription. Reciprocally, forced expression of Klf4 partially recovered the odontoblastic differentiation capacity of mDPCs with Mycn knockdown.ConclusionsOur results elucidated that mesenchymal Mycn modulates the odontoblastic commitment of dental papilla cells by directly regulating Klf4. Our study illustrated the role of Mycn in dentin development and furthers our general comprehension of the transcription factor networks involved in the dentinogenesis process. Thus, these results may provide new insight into dentin hypoplasia and bioengineered dentin regeneration.

CircKLF4 Upregulates Klf4 and Endoglin to Promote Odontoblastic Differentiation of Mouse Dental Papilla Cells via Sponging miRNA-1895 and miRNA-5046.

circular RNAs (circRNAs) is a broad and diverse endogenous subfamily of non-coding RNAs, regulating the gene expression by acting as a microRNA (miRNA) sponge. However, the biological functions of circRNAs in odontoblast differentiation remain largely unknown. Our preliminary study identified an unknown mouse circRNA by circRNA sequencing generated from mouse dental papilla and we termed it circKLF4. In this study, quantitative real-time PCR and in situ hybridization were used and demonstrated that circKLF4 was upregulated during odontoblastic differentiation. Gene knockdown and overexpression assays indicated that circKLF4 promoted odontoblastic differentiation of mouse dental papilla cells (mDPCs). Mechanistically, we found that circKLF4 increased the linear KLF4 expression in a microRNA-dependent manner. By mutating the binding sites of microRNA and circKLF4, we further confirmed that circKLF4 acted as sponge of miRNA-1895 and miRNA-5046 to promote the expression of KLF4. We then also found that ENDOGLIN was also up-regulated by circKLF4 by transfection of circKLF4 overexpression plasmids with or without microRNA inhibitor. In conclusion, circKLF4 increases the expression of KLF4 and ENDOGLIN to promote odontoblastic differentiation via sponging miRNA-1895 and miRNA-5046.

Biocompatibility and pro-mineralization effect of tristrontium aluminate cement for endodontic use

Background/purposeTristrontium aluminate (S3A) is a hydraulic cement with setting behavior similar to that of mineral trioxide aggregate (MTA). This study examined the biological effects of S3A on mouse dental papilla cells (MDPs) in vitro and on rat exposed pulps in vivo. Materials and methodsExtracts of S3A and MTA were prepared by immersing each cement in ultrapure water. MDPs were cultured with S3A or MTA extracts, and cell proliferation was evaluated with a tetrazolium-salt assay. Attachment of MDPs on the set cements was examined with scanning electron microscopy (SEM). mRNA expression of bone morphogenic protein (Bmp2), osteocalcin (Oc) and osteopontin (Opn) in MDPs exposed to S3A or MTA extracts was determined with reverse transcription-quantitative polymerase chain reaction. Mineralized nodule formation was evaluated with Alizarin Red S staining. Simulated body fluid (SBF)-dipped S3A was examined with SEM and energy dispersive X-ray analysis (EDX). Exposed molar pulps of male Wistar rats capped with S3A or MTA were histologically examined. ResultsS3A extract did not inhibit proliferation of MDPs. Set S3A and MTA exhibited attachment of MDPs on their surface. S3A extract showed significantly higher mineralized nodule formation and mRNA expression of Bmp2, Oc, and Opn than did MTA extract. SBF-dipped S3A exhibited formation of surface precipitates, which were composed of Ca, P, Sr, and Al. Direct pulp capping with S3A and with MTA induced mineralized tissue repair of the exposed pulp. ConclusionS3A possesses biocompatibility and pro-mineralization effects comparable to those of MTA.

Differential lncRNA/mRNA Expression Profiling and Functional Network Analyses in Bmp2 Deletion of Mouse Dental Papilla Cells.

Bmp2 is essential for dentin development and formation. Bmp2 conditional knock-out (KO) mice display a similar tooth phenotype of dentinogenesis imperfecta (DGI). To elucidate a foundation for subsequent functional studies of cross talk between mRNAs and lncRNAs in Bmp2-mediated dentinogenesis, we investigated the profiling of lncRNAs and mRNAs using immortalized mouse dental Bmp2 flox/flox (iBmp2fx/fx) and Bmp2 knock-out (iBmp2ko/ko) papilla cells. RNA sequencing was implemented to study the expression of the lncRNAs and mRNAs. Quantitative real-time PCR (RT-qPCR) was used to validate expressions of lncRNAs and mRNAs. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to predict functions of differentially expressed genes (DEGs). Protein–protein interaction (PPI) and lncRNA–mRNA co-expression network were analyzed by using bioinformatics methods. As a result, a total of 22 differentially expressed lncRNAs (16 downregulated vs 6 upregulated) and 227 differentially expressed mRNAs (133 downregulated vs. 94 upregulated) were identified in the iBmp2ko/ko cells compared with those of the iBmp2fx/fx cells. RT-qPCR results showed significantly differential expressions of several lncRNAs and mRNAs which were consistent with the RNA-seq data. GO and KEGG analyses showed differentially expressed genes were closely related to cell differentiation, transcriptional regulation, and developmentally relevant signaling pathways. Moreover, network-based bioinformatics analysis depicted the co-expression network between lncRNAs and mRNAs regulated by Bmp2 in mouse dental papilla cells and symmetrically analyzed the effect of Bmp2 during dentinogenesis via coding and non-coding RNA signaling.

Chromatin Accessibility Predetermines Odontoblast Terminal Differentiation

Embryonic development and stem cell differentiation are orchestrated by changes in sequential binding of regulatory transcriptional factors to their motifs. These processes are invariably accompanied by the alternations in chromatin accessibility, conformation, and histone modification. Odontoblast lineage originates from cranial neural crest cells and is crucial in dentinogenesis. Our previous work revealed several transcription factors (TFs) that promote odontoblast differentiation. However, it remains elusive as to whether chromatin accessibility affects odontoblast terminal differentiation. Herein, integration of single-cell RNA-seq and bulk RNA-seq revealed that in vitro odontoblast differentiation using dental papilla cells at E18.5 was comparable to the crown odontoblast differentiation trajectory of OC (osteocalcin)-positive odontogenic lineage. Before in vitro odontoblast differentiation, ATAC-seq and H3K27Ac CUT and Tag experiments demonstrated high accessibility of chromatin regions adjacent to genes associated with odontogenic potential. However, following odontoblastic induction, regions near mineralization-related genes became accessible. Integration of RNA-seq and ATAC-seq results further revealed that the expression levels of these genes were correlated with the accessibility of nearby chromatin. Time-course ATAC-seq experiments further demonstrated that odontoblast terminal differentiation was correlated with the occupation of the basic region/leucine zipper motif (bZIP) TF family, whereby we validated the positive role of ATF5 in vitro. Collectively, this study reports a global mapping of open chromatin regulatory elements during dentinogenesis and illustrates how these regions are regulated via dynamic binding of different TF families, resulting in odontoblast terminal differentiation. The findings also shed light on understanding the genetic regulation of dentin regeneration using dental mesenchymal stem cells.

Expression of nitric oxide synthases in rat odontoblasts and the role of nitric oxide in odontoblastic differentiation of rat dental papilla cells.

As precursor cells of odontoblasts, dental papilla cells (DPCs) form the dentin-pulp complex during tooth development. Nitric oxide (NO) regulates the functions of multiple cells and organ tissues, including stem cell differentiation and bone formation. In this paper, we explored the involvement of NO in odontoblastic differentiation. We verified the expression of NO synthase (NOS) in rat odontoblasts by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining and immunohistochemistry in vivo. The expression of all three NOS isoforms in rat DPCs was confirmed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting in vitro. The expression of neuronal NOS and endothelial NOS was upregulated during the odontoblastic differentiation of DPCs. Inhibition of NOS function by NOS inhibitor l-NG -monomethyl arginine (L-NMMA) resulted in reduced formation of mineralized nodules and expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein (DMP1) during DPC differentiation. The NO donor S-nitroso-N-acetylpenicillamine (SNAP, 0.1, 1, 10, and 100μM) promoted the viability of DPCs. Extracellular matrix mineralization and odontogenic markers expression were elevated by SNAP at low concentrations (0.1, 1, and 10μM) and suppressed at high concentration (100μM). Blocking the generation of cyclic guanosine monophosphate (cGMP) with 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ) abolished the positive influence of SNAP on the odontoblastic differentiation of DPCs. These findings demonstrate that NO regulates the odontoblastic differentiation of DPCs, thereby influencing dentin formation and tooth development.

Periostin in Periodontics: A Brief Insight

The prime objective of the periodontal treatment is to re-establish the lost periodontal tissue by forming new attachment of periodontal ligament, cementum and the alveolar bone. Periostin is a matricellular protein with its structure closely resembling to that of Drosophila fasciclin. It is expressed in the periodontal ligament, periosteum, alveolar bone, adipose tissue as well as skeletal tissue and has a key role in role in tooth development. This protein molecule has a striking similarity to the molecule beta ig-h3 which is induced by transforming growth factor beta (TGF-beta), promotes the adhesion and spreading of fibroblasts and aids in collagen formation. It is possibly involved in the post surgical regeneration of periodontal hard and soft tissues. Periostin is also linked to dental papilla cells and is associated with hard tissue formation stages of tooth development. This mini review focuses on the various aspects of periostin related to maintenance and development of collagen rich connective tissues around the teeth. Keywords: Periostin, Periodontal ligament, Periodontal regeneration, Alveolar bone.

C-Jun N-terminal kinase (JNK) pathway activation is essential for dental papilla cells polarization.

During tooth development, dental papilla cells differentiate into odontoblasts with polarized morphology and cell function. Our previous study indicated that the C-Jun N-terminal kinase (JNK) pathway regulates human dental papilla cell adhesion, migration, and formation of focal adhesion complexes. The aim of this study was to further examine the role of the JNK pathway in dental papilla cell polarity formation. Histological staining, qPCR, and Western Blot suggested the activation of JNK signaling in polarized mouse dental papilla tissue. After performing an in vitro tooth germ organ culture and cell culture, we found that JNK inhibitor SP600125 postponed tooth germ development and reduced the polarization, migration and differentiation of mouse dental papilla cells (mDPCs). Next, we screened up-regulated polarity-related genes during dental papilla development and mDPCs or A11 differentiation. We found that Prickle3, Golga2, Golga5, and RhoA were all up-regulated, which is consistent with JNK signaling activation. Further, constitutively active RhoA mutant (RhoA Q63L) partly rescued the inhibition of SP600125 on cell differentiation and polarity formation of mDPCs. To sum up, this study suggests that JNK signaling has a positive role in the formation of dental papilla cell polarization.

SIRT4 regulates rat dental papilla cell differentiation by promoting mitochondrial functions.

SIRT4 is a mitochondrial sirtuin. Owing to its dependance on the cofactor nicotinamide adenine dinucleotide (NAD+), SIRT4 can act as a mitochondrial metabolic sensor of cellular energy status. We have previously shown that enhancement of mitochondrial functions is vital for the odontogenic diff ;erentiation of dental papilla cells (DPCs) during dentinogenesis. However, whether SIRT4 serves as an effective regulator of DPC diff ;erentiation by affecting mitochondrial functions remains unexplored. Primary DPCs obtained from the first molar dental papilla of neonatal Sprague-Dawley rats were used in this study. The expression pattern of SIRT4 was observed by immunohistochemistry in the first molar of postnatal day 1 (P1) rats. The changes in SIRT4 expression during odontogenic DPC differentiation were evaluated using real-time quantitative polymerase chain reaction (PCR), western blotting, and immunofluorescence. DPCs with loss (small interfering RNA-mediated knockdown) and gain (plasmid transfection-induced overexpression) of SIRT4 function were used to explore the role of SIRT4 in odontogenic differentiation. Mitochondrial function assays were performed using ATP, reactive oxygen species (ROS), and NAD+/NADH kits to investigate the potential mechanisms involved in SIRT4-mediated dentinogenesis. In the present study, we found that SIRT4 expression increased in a time-dependent manner during odontogenic differentiation bothin vivo and in vitro. Sirt4 knockdown resulted in reduced odontogenic differentiation and mineralization, whereas an opposite effect was observed with SIRT4 overexpression. Furthermore, our results verified that in addition to reducing DPC differentiation, Sirt4 knockdown could also significantly reduce ATP levels, elevate the NAD+/NADH ratio, and increase ROS levels. SIRT4 regulates mitochondrial functions and the antioxidant capacity of DPCs, thereby influencing dentin formation and tooth development, a phenomenon that may provide a foundation for better understanding the specific molecular mechanisms underlying dentin regeneration.

RORα Regulates Odontoblastic Differentiation and Mediates the Pro-Odontogenic Effect of Melatonin on Dental Papilla Cells.

Dental papilla cells (DPCs), precursors of odontoblasts, are considered promising seed cells for tissue engineering. Emerging evidence suggests that melatonin promotes odontoblastic differentiation of DPCs and affects tooth development, although the precise mechanisms remain unknown. Retinoid acid receptor-related orphan receptor α (RORα) is a nuclear receptor for melatonin that plays a critical role in cell differentiation and embryonic development. This study aimed to explore the role of RORα in odontoblastic differentiation and determine whether melatonin exerts its pro-odontogenic effect via RORα. Herein, we observed that RORα was expressed in DPCs and was significantly increased during odontoblastic differentiation in vitro and in vivo. The overexpression of RORα upregulated the expression of odontogenic markers, alkaline phosphatase (ALP) activity and mineralized nodules formation (p < 0.05). In contrast, odontoblastic differentiation of DPCs was suppressed by RORα knockdown. Moreover, we found that melatonin elevated the expression of odontogenic markers, which was accompanied by the upregulation of RORα (p < 0.001). Utilising small interfering RNA, we further demonstrated that RORα inhibition attenuated melatonin-induced odontogenic gene expression, ALP activity and matrix mineralisation (p < 0.01). Collectively, these results provide the first evidence that RORα can promote odontoblastic differentiation of DPCs and mediate the pro-odontogenic effect of melatonin.

Effects of transforming growth factor-β1 on odontoblastic differentiation in dental papilla cells is determined by IPO7 expression level

Transforming growth factor β1 (TGF-β1) is one of the broad-spectrum growth-promoting factors that participate in tooth development. The influence of TGF-β1 on the odontoblastic differentiation is still controvercy. Mouse primary dental papilla cells (mDPCs) as well as an immortalized mouse dental papilla cell line (mDPC6Ts) were treated with exogenous TGF-β1 during odontoblastic differentiation. RT-qPCR, Western blot, alizarin red staining and ALP staining were carried out to investigate the influence of TGF-β1 on odontoblastic differentiation. IPO7, important for SMAD complex translocation was also detected in mDPCs and mDPC6Ts in response to TGF-β1. After silencing IPO7 by transfection, the translocation process of P-SMAD2 was investigated by nuclear and cytoplasmic extraction as well as co-immunoprecipitation assay. The odontogenic markers, mineralization and IPO7 expression were significantly up-regulated in TGF-β1-treated mDPCs while down-regulated in mDPC6Ts. The total level of P-SMAD2 was not influenced by IPO7 in mDPCs, however, IPO7 could bind to P-SMAD2 and affect the nuclear-cytoplasm-shuttling of P-SMAD2. Our data demonstrated that TGF-β1 plays opposite roles in odontoblast differentiation in mDPCs and immortalized mouse dental papilla cell line (mDPC6Ts), which is determined by IPO7.

Técnica de revascularización en odontopediatría paso a paso. A propósito de un caso

Introducción: La revascularización es un procedimiento actual para dientes necróticos con ápice inmaduro; supone una opción de manejo clínico más conservador con respecto a los tratamientos tradicionales de apicoformación, en el cual, por medio de la inducción de las células madre de la papila dental periapical, se crea un sangrado dentro del conducto radicular, el cual se convierte en un coágulo estable que permite la maduración fisiológica radicular, a la vez que el engrosamiento de las paredes del conducto. El objetivo de este tratamiento es permitir un cierre completo del ápice. Caso clínico: Se presenta el caso de un niño de 7 años de edad con abceso apical en el diente 21, se realiza un tratamiento de revascularización siguiendo la técnica descrita por Hoshino (1996) de apósitos intraconducto con alguna modificación. Siguiendo la teoría descrita por Iwaya en 2001, y Banchs y Trope en 2004, el canal casose desinfecta con abundante irrigación y se rellena con una combinación de pasta bi-antibiótica. Una vez que se completa el protocolo de desinfección, el ápice se irrita mecánicamente para iniciar el sangrado en el canal y producir un coágulo sanguíneo hasta el nivel de la unión cemento-esmalte, colocando Agregado Trióxido Mineral como barrera entre el coágulo y la restauración final. En este caso, la combinación de un canal desinfectado, una matriz en la que podría crecer tejido nuevo y un sello coronal efectivo parece producir el ambiente necesario para una revascularización exitosa. Conclusiones: Bajo las circunstancias de este caso, se puede concluir que el protocolo del tratamiento seguido (regeneración apical y tapón de MTA) es un tratamiento de éxito clínico para conseguir el cierre apical en dientes inmaduros. Este éxito también dependerá de variables como el material de desinfección radicular: el Cloruro Sódico seguido de una solución salina es capaz de desinfectar los conductos sin tener que instrumentar las débiles paredes dentinarias. El uso de pasta bi-antibiótica intracanal utilizada en el caso clínico previene de la pigmentación dental que provocan las tetraciclinas cuando se utilizan en la pasta tri-antibiótica. Se necesitan revisiones de control radiográfico cada 3 meses el primer año y anualmente hasta los 4 años de realizada la revascularización, para comprobar el éxito del tratamiento. Actualmente la regeneración tisular está enfocada hacia el desarrollo de sistemas de cultivo que permitan reproducir células embrionarias hacia tejidos dentales perdidos.