Disrupted odontoblast differentiation and dentin dysplasia in Epiprofin-deficient mice.

Tooth formation is a process tightly regulated by reciprocal interactions between epithelial and mesenchymal tissues. These epithelial-mesenchyme interactions regulate the expression of target genes via transcription factors. Among the regulatory elements governing this process, Epiprofin/Sp6 is a zinc finger transcription factor which is expressed in the embryonic dental epithelium and in differentiating pre-odontoblasts. Epiprofin knockout (Epfn-/-) mice present severe dental abnormalities, such as supernumerary teeth and enamel hypoplasia. Here, we describe dentin defects in molars and incisors of Epfn-/- mice. We observed that in the absence of Epfn, markers of early odontoblast differentiation, such as alkaline phosphatase activity, Dsp/Dpp expression, and Collagen Type I deposition, are downregulated. In addition, the expression of tight and gap junction proteins was severely impaired in the predontoblastic cell layer of developing Epfn-/- molars. Altogether, our data shows that Epfn is crucial for the proper differentiation of dental mesenchymal cells towards functional odontoblasts and subsequent dentin-matrix deposition.

[Inhibition of connexin 43-mediated hemichannel activity promotes odontoblast differentiation of human dental pulp cells induced by lipopolysaccharide].

PURPOSE: To investigate the role and mechanism of connexin 43（Cx43）in odontoblast differentiation of human dental pulp cells (hDPCs) induced by lipopolysaccharide (LPS). METHODS: The maxillary first molar injury model of SD rats was established. The expression pattern of Cx43 in dental pulp repair after injury was detected by immunofluorescence(IF) staining. hDPCs was respectively stimulated with 0, 1, 10, 100 and 1 000 ng/mL LPS for 6 h to screen the optimal concentration, and then the expression of Cx43 was inhibited and overexpressed in hDPCs. Quantitative real-time PCR(qRT-PCR) and Western blot(WB) were used to detect the expression of Cx43 and dentin sialophosphoprotein (DSPP), dental matrix protein-1 (DMP-1), osterix (Osx) and extracellular signal-regulated kinase (ERK) activity. Furthermore, hDPCs were treated with specific Cx43 channel inhibitors to investigate the effect of Cx43-mediated channel activity in odontoblast differentiation of hDPCs, and to explore the role and mechanism of Cx43 in regulating odontoblast differentiation of hDPCs induced by LPS. Statistical analysis was performed with SPSS 26.0 software package. RESULTS: IF results showed that Cx43 was mainly expressed in the odontoblast layer in healthy dental pulp tissues. At 3-24 h after tooth injury, the expression of Cx43 decreased and then gradually increased to the normal level; from 3 days to 2 weeks after injury, the expression of Cx43 tended to be down-regulated which was in the odontoblast layer and pulp proper. The expression of DSPP mRNA was significantly up-regulated in the hDPCs stimulated with 10 ng/mL LPS for 6 h(P＜0.01). Inhibition of Cx43 significantly up-regulated the expression of DSPP, DMP-1 and Osx mRNA induced by LPS in hDPCs(P＜0.05), while overexpression of Cx43 obviously inhibited the expression of factors related to LPS-induced odontoblast differentiation(P＜0.01) and the fluorescence intensity of DSPP. 10 ng/mL LPS activated ERK signal in hDPCs, and overexpression of Cx43 significantly attenuated the activity of ERK signal induced by LPS(P＜0.01). Inhibition of Cx43-mediated hemichannel (HC) promoted mRNA expression of factors related to odontoblast differentiation in hDPCs and the activity of ERK signal induced by LPS(P＜0.05), while blocking Cx43-mediated gap junction channel (GJC) inhibited odontoblast differentiation. CONCLUSIONS: Cx43 participates in the regulation of dental pulp repair after injury, and its expression shows a downward trend as a whole. Inhibition of Cx43 or blocking of HC promotes LPS-induced ERK signal activity and odontoblast differentiation of hDPCs.

Effect of NAMPT on the proliferation and apoptosis in odontoblast-like MDPC-23 cell.

This study aimed to evaluate the physiological role of NAMPT associated with MDPC-23 odontoblast cell proliferation. Cell viability was measured using the (DAPI) staining, caspase activation analysis and immunoblotting were performed. Visfatin promoted MDPC-23 odontoblast cell growth in a dose-dependent manner. Furthermore, the up-regulation of Visfatin promoted odontogenic differentiation and accelerated mineralization through an increase in representative odontoblastic biomarkers in MDPC-23 cells. However, FK-866 cell growth in a dose-dependent manner induced nuclear condensation and fragmentation. FK-866-treated cells showed H&E staining and increased apoptosis compared to control cells. The expression of anti-apoptotic factors components of the mitochondria-dependent intrinsic apoptotic pathway significantly decreased following FK-866 treatment. The expression of pro-apoptotic increased upon FK-866 treatment. In addition, FK-866 activated caspase-3 and PARP to induce cell death. In addition, after treating FK-866 for 72 h, the 3/7 activity of MDPC-23 cells increased in a concentration-dependent manner, and the IHC results also confirmed that Caspase-3 increased in a concentration-dependent. Therefore, the presence or absence of NAMPT expression in dentin cells was closely related to cell proliferation and formation of extracellular substrates.

Peptide KN-17-Loaded Supramolecular Hydrogel Induces the Regeneration of the Pulp-Dentin Complex.

Regenerating the pulp-dentin complex remains a decisive factor during apexification for immature permanent teeth. Peptide KN-17, which was modified based on the structure of cecropin B, could effectively interfere with bacterial growth and induce the migration of human bone marrow stromal cells (hBMSCs). This study aimed to investigate the effect of KN-17 on the tissue regeneration. To our surprise, KN-17 can significantly stimulate angiogenesis in vitro and in vivo, which may provide a guarantee for apical closure. Herein, a novel peptide/KN-17 coassembled hydrogel is developed via a heating-cooling process. Npx-FFEY/KN-17 supramolecular hydrogel can induce vessel development, stimulate odontogenic differentiation of human dental pulp stem cells (hDPSCs), and exert an antibacterial effect on Enterococcus faecalis (E. faecalis). Furthermore, coronal pulp excised rat molars are supplied with KN-17 or KN-17-loaded hydrogel and transplanted subcutaneously in BALB/c-nu mice. After 4 weeks, the hydrogel Npx-FFEY/KN-17 stimulates the formation of multiple odontoblast-like cells and dentin-like structures. Our findings demonstrate that the KN-17-loaded hydrogel can promote the regeneration of the pulp-dentin complex for continued root development.

Anti-inflammatory cerium-containing nano-scaled mesoporous bioactive glass for promoting regenerative capability of dental pulp cells.

AIMS: This study aimed to investigate the anti-inflammatory and odontoblastic effects of cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBGNs) on dental pulp cells as novel pulp-capping agents. METHODOLOGY: Ce-MBGNs were synthesized using a post-impregnation strategy based on the antioxidant properties of Ce ions and proposed the first use of Ce-MBGNs for pulp-capping application. The biocompatibility of Ce-MBGNs was analysed using the CCK-8 assay and apoptosis detection. Additionally, the reactive oxygen species (ROS) scavenging ability of Ce-MBGNs was measured using the 2,7-Dichlorofuorescin Diacetate (DCFH-DA) probe. The anti-inflammatory effect of Ce-MBGNs on THP-1 cells was further investigated using flow cytometry and quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, the effect of Ce-MBGNs on the odontoblastic differentiation of the dental pulp cells (DPCs) was assessed by combined scratch assays, RT-qPCR, western blotting, immunocytochemistry, Alizarin Red S staining and tissue-nonspecific alkaline phosphatase staining. Analytically, the secretions of tumour necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were detected with enzyme-linked immunosorbent assay (ELISA). RESULTS: Ce-MBGNs were confirmed to effectively scavenge ROS in THP-1-derived macrophages and DPCs. Flow cytometry and RT-qPCR assays revealed that Ce-MBGNs significantly inhibited the M1 polarization of macrophages (Mφ). Furthermore, the protein levels of TNF-α and IL-1ß were downregulated in THP-1-derived macrophages after stimulation with Ce-MBGNs. With a step-forward virtue of promoting the odontoblastic differentiation of DPCs, we further confirmed that Ce-MBGNs could regulate the formation of a conductive immune microenvironment with respect to tissue repair in DPCs, which was mediated by macrophages. CONCLUSIONS: Ce-MBGNs protected cells from self-produced oxidative damage and exhibited excellent immunomodulatory and odontoblastic differentiation effects on DPCs. As a pulp-capping agent, this novel biomaterial can exert anti-inflammatory effects and promote restorative dentine regeneration in clinical treatment. We believe that this study will stimulate further correlative research on the development of advanced pulp-capping agents.

Hyaluronan degradation by HYAL2 is essential for odontoblastic differentiation and migration of mouse dental papilla cells.

The coordination between odontoblastic differentiation and directed cell migration of mesenchymal progenitors is necessary for regular dentin formation. The synthesis and degradation of hyaluronan (HA) in the extracellular matrix create a permissive niche that directly regulates cell behaviors. However, the role and mechanisms of HA degradation in dentin formation remain unknown. In this work, we present that HA digestion promotes odontoblastic differentiation and cell migration of mouse dental papilla cells (mDPCs). Hyaluronidase 2 (HYAL2) is responsible for promoting odontoblastic differentiation through degrading HA, while hyaluronidase 1 (HYAL1) exhibits negligible effect. Silencing Hyal2 generates an extracellular environment rich in HA, which attenuates F-actin and filopodium formation and in turn inhibits cell migration of mDPCs. In addition, activating PI3K/Akt signaling significantly rescues the effects of HA accumulation on cytodifferentiation. Taken together, the results confirm the contribution of HYAL2 to HA degradation in dentinogenesis and uncover the mechanism of the HYAL2-mediated HA degradation in regulating the odontoblastic differentiation and migration of mDPCs.

TFEB regulates the odontoblastic differentiation of dental pulp stem cells by promoting a positive feedback loop between mitophagy and glycolysis.

OBJECTIVE: To evaluate the regulatory effect of transcription factor EB (TFEB) on the odontoblastic differentiation of dental pulp stem cells(DPSCs) in vivo and in vitro. DESIGNS: RNA-seq was used to detect differentially expressed genes in differentiated DPSCs. Lysosomes and the expression of the related gene TFEB were examined in DPSCs. DPSCs were then transfected with lentivirus for TFEB-overexpression. Cell proliferation was detected using CCK-8 and EdU assays, while cell differentiation was detected using ALP and ARS detection kits. Subsequently, mitophagy and cell metabolism were examined using TEM and Seahorse. An odontoblastic differentiation model was constructed subcutaneously in nude mice. Finally, the effects of glycolysis and mitophagy inhibitors were evaluated on odontoblastic differentiation and the associated mechanisms were explored. RESULTS: TFEB overexpression promoted a significant increase in ALP activity and the expression of differentiation-related genes in DPSCs, while it inhibited cell proliferation. In vivo, TFEB overexpression caused higher bone volume/trabecular volume(BV/TV), and an increase in collagen formation and heightened DMP-1 expression. Furthermore, Seahorse flux analysis demonstrated that TFEB promoted metabolic reprogramming. Transmission electron microscope(TEM) results indicated an increase in mitochondrial autophagosomes after TFEB overexpression, and the expression of mitophagy-related genes was also elevated. The odontoblastic differentiation of DPSCs promoted by TFEB overexpression was suppressed after the addition of 2-DG and Midiv-1. Addition of Midiv-1 reduced the glycolytic rate of DPSCs, while addition of 2-DG also decreased the mitophagy level of the cells. CONCLUSIONS: Our results showed that TFEB promoted the odontoblastic differentiation of DPSCs and identified mitophagy and metabolic reprogramming as a positive feedback loop.

The Reparative Function of MMP13 in Tertiary Reactionary Dentinogenesis after Tooth Injury.

MMP13 gene expression increases up to 2000-fold in mineralizing dental pulp cells (DPCs), with research previously demonstrating that global MMP13 deletion resulted in critical alterations in the dentine phenotype, affecting dentine-tubule regularity, the odontoblast palisade, and significantly reducing the dentine volume. Global MMP13-KO and wild-type mice of a range of ages had their molar teeth injured to stimulate reactionary tertiary dentinogenesis. The response was measured qualitatively and quantitatively using histology, immunohistochemistry, micro-CT, and qRT-PCR in order to assess changes in the nature and volume of dentine deposited as well as mechanistic links. MMP13 loss affected the reactionary tertiary dentine quality and volume after cuspal injury and reduced Nestin expression in a non-exposure injury model, as well as mechanistic links between MMP13 and the Wnt-responsive gene Axin2. Acute pulpal injury and pulp exposure to oral fluids in mice teeth showed upregulation of the MMP13 in vivo, with an increase in the gene expression of Mmp8, Mmp9, and Mmp13 evident. These results indicate that MMP13 is involved in tertiary reactionary dentine formation after tooth injury in vivo, potentially acting as a key molecule in the dental pulp during dentine-pulp repair processes.

NOTUM plays a bidirectionally modulatory role in the odontoblastic differentiation of human stem cells from the apical papilla through the WNT/ß-catenin signaling pathway.

OBJECTIVE: Notum is a secreted deacylase, which is crucial for tooth dentin development in mice. This study aimed to investigate the effect of NOTUM on the odontoblastic differentiation of human stem cells from the apical papilla (hSCAPs), to reveal the potential value of NOTUM in pulp-dentin complex regeneration. DESIGN: The expression pattern of NOTUM in human tooth germs and during in vitro odontoblastic differentiation of hSCAPs was evaluated by immunohistochemical staining, and quantitative polymerase chain reaction, respectively. To manipulate the extracellular NOTUM level, ABC99 or small interfering RNA was used to down-regulate it, while recombinant NOTUM protein was added to up-regulate it. The effects of changing NOTUM level on the odontoblastic differentiation of hSCAPs and its interaction with the WNT/ß-catenin signaling pathway were studied using alkaline phosphatase staining, alizarin red staining, quantitative polymerase chain reaction, and western blot. RESULTS: NOTUM was observed in the apical papilla of human tooth germs. During in vitro odontoblastic differentiation of hSCAPs, NOTUM expression initially increased, while the WNT/ß-catenin pathway was activated. Downregulation of NOTUM hindered odontoblastic differentiation. Recombinant NOTUM protein had varying effects on odontoblastic differentiation depending on exposure duration. Continuous addition of the protein inhibited both odontoblastic differentiation and the WNT/ß-catenin pathway. However, applying the protein solely in the first 3 days enhanced odontoblastic differentiation and up-regulated the WNT/ß-catenin pathway. CONCLUSION: NOTUM demonstrated a bidirectional impact on in vitro odontoblastic differentiation of hSCAPs, potentially mediated by the WNT/ß-catenin pathway. These findings suggest its promising potential for pulp-dentin complex regeneration.

Role of YAP in Odontoblast Damage Repair in a Dentin Hypersensitivity Model.

OBJECTIVES: The aim of this study was to investigate the molecular mechanism underlying odontoblast damage repair in dentin hypersensitivity (DH) and the role of Yes-associated protein (YAP) in this process. METHODS: The DH model was constructed in Sprague-Dawley (SD) rats, and the in vivo expression of Piezo1, Integrin αvß3, YAP, and dentin sialophosphoprotein (DSPP) was detected by immunohistochemistry. COMSOL Multiphysics software was used to simulate the dentinal tubule fluid flow velocity and corresponding fluid shear stress (FSS) on the odontoblast processes. MDPC-23 cells were cultured in vitro and loaded with a peristaltic pump for 1 hour at FSS values of 0.1, 0.3, 0.5, and 0.7 dyne/cm2. The expression of Piezo1, Integrin αvß3, and YAP was detected by immunofluorescence. Verteporfin (a YAP-specific inhibitor) was utilised to confirm the effect of YAP on the expression of dentineogenesis-related protein under FSS. RESULTS: The level and duration of external mechanical stimuli have an effect on the functional expression of odontoblasts. In DH, the harder the food that is chewed, the faster the flow of the dentinal tubule fluid and the greater the FSS on the odontoblast processes. The expression of Piezo1, Integrin αvß3, and YAP can be promoted when the FSS is less than 0.3 dyne/cm2. After YAP inhibition, the DSPP protein expression level was reduced at 0.3 dyne/cm2 FSS. CONCLUSIONS: These results suggest that appropriate FSS can enhance the expression of odontoblast-related factors in odontoblasts via the Piezo1-Integrin αvß3-YAP mechanotransduction pathway and the YAP appears to play an essential role in the response of odontoblasts to external mechanical stimuli.

Nicotinamide Riboside Modulates HIF-1 Signaling to Maintain and Enhance Odontoblastic Differentiation in Human Dental Pulp Stem Cells.

Human dental pulp stem cells (hDPSCs) play a vital role in the regeneration of the pulp-dentin complex after pulp disease. While the regeneration efficiency relies on the odontoblastic differentiation capacity of hDPSCs, this is difficult to regulate within the pulp cavity. Although nicotinamide riboside (NR) has been found to promote tissue regeneration, its specific role in pulp-dentin complex regeneration is not fully understood. Here, we aimed to explore the role of NR in the odontoblastic differentiation of hDPSCs and its underlying molecular mechanism. It was found that NR enhanced the viability and retarded senescence in hDPSCs with higher NAD+/NADH levels. In contrast to the sustained action of NR, the multi-directional differentiation of hDPSCs was enhanced after NR pre-treatment. Moreover, in an ectopic pulp regeneration assay in nude mice, transplantation of hDPSCs pretreated with NR promoted the formation of a dentin-like structure surrounded by cells positively expressing DMP-1 and DSPP. RNA-Seq demonstrated inhibition of the HIF-1 signaling pathway in hDPSCs pretreated with NR. The number of HIF-1α-positive cells was significantly decreased in hDPSCs pretreated by NR in vivo. Similarly, NR significantly downregulated the expression of HIF-1α in vitro. The findings suggested that NR could potentially regulate hDPSC odontoblastic differentiation and promote the development of innovative strategies for dental pulp repair.

Piezo Mediates the Mechanosensation and Injury-Repair of Pulpo-Dentinal Complex.

OBJECTIVES: The aim of this research was to investigate the functions of Piezo channels in dentin defect, including mechanical signalling and odontoblast responses. METHODS: Rat dentin-defect models were constructed, and spatiotemporal expression of Piezo proteins was detected in the pulpo-dentinal complex. Real-time polymerase chain reaction (rtPCR) was used to investigate the functional expression pattern of Piezo channels in odontoblasts. Moreover, RNA interference technology was employed to uncover the underlying mechanisms of the Piezo-driven inflammatory response and repair under fluid shear stress (FSS) conditions in vitro. RESULTS: Piezo1 and Piezo2 were found to be widely expressed in the odontoblast layer and dental pulp in the rat dentin-defect model during the end stage of reparative dentin formation. The expression levels of the Piezo1 and Piezo2 genes in MDPC-23 cells were high in the initial stage under FSS loading and then decreased over time. Moreover, the expression trends of inflammatory, odontogenic, and mineralisation genes were generally contrary to those of Piezo1 and Piezo2 over time. After silencing of Piezo1/Piezo2, FSS stimulation resulted in significantly higher expression of inflammatory, odontogenesis, and mineralisation genes in MDPC-23 cells. Finally, the expression of genes involved in the integrin ß1/ERK1 and Wnt5b/ß-catenin signalling pathways was changed in response to RNA silencing of Piezo1 and Piezo2. CONCLUSIONS: Piezo1 and Piezo2 may be involved in regulating the expression of inflammatory and odontogenic genes in odontoblasts stimulated by FSS.

The effect of Par3 on the cellular junctions and biological functions of odontoblast-lineage cells.

Perfect intercellular junctions are key for odontoblast barrier function. However, whether Partitioning defective-3 (Par3) is expressed in odontoblasts and its potential effects on odontoblast junctions are unknown. Herein, we investigated the effect of Par3 on cellular junctions and the biological behavior of odontoblast-lineage cells (OLCs). Whole-transcriptome sequencing was used to analyze the effects of Par3 on OLCs and the underlying molecular mechanism. Par3 was detected under physiological and inflammatory conditions in OLCs. To investigate the regulatory effect of Par3 on junctions between mouse OLCs, the effects of Par3 downregulation on the proliferation, migration, cycle and apoptosis of OLCs were detected by 5-ethyl-2'-deoxyuridine (EdU) and Transwell assays and flow cytometry. Western blotting and alizarin red S and alkaline phosphatase (ALP) staining were used to observe the effect of Par3 downregulation on OLC mineralization. Whole-transcriptome sequencing was used to investigate the biological role of Par3 in OLCs and potential molecular mechanisms. Par3 was located along the odontoblast layer in the rat pulp tissue and in the cytoplasm of OLCs. Par3 expression was downregulated under inflammatory conditions. The OLC junctions were discontinuous, and total Zona occluden-1 (ZO-1) expression and expression of ZO-1 at the membrane in OLCs were reduced after Par3 silencing (P < 0.05). Expression of a junction-related protein (ZO-1) was downregulated after the downregulation of Par3 (P < 0.05), and ZO-1 moved from the cell membrane to the cytoplasm. OLC proliferation and migration were enhanced, but apoptosis and mineralization were inhibited in shPar3-transfected cells (P < 0.05). Sequencing identified 2996 differentially expressed genes (DEGs), which were mainly enriched in the response to stimuli and binding. Downregulation of Par3 could overactivate the PI3k-AKT pathway by promoting AKT phosphorylation (P < 0.05). Downregulation of Par3 may disrupt junctions between OLCs by affecting ZO-1 expression and distribution and promote OLC proliferation and migration but inhibit OLC mineralization. Par3 may interact with 14-3-3 proteins for PI3K-AKT pathway activation to affect OLC junctions and function.

The effect of a soft diet on molar dentin formation during the occlusal establishment period.

OBJECTIVE: This study intends to investigate the effect of a soft food diet on molar dentin formation during the occlusal establishment period. It can provide dietary guidance for infants to strengthen their dental structure. DESIGN: 60 BALB/c mice were used to obtain mandibles during lactation (P0.5, P7.5, P15.5, P21.5) and occlusal establishment (P27.5, P33.5, P60.5). The mice were randomly divided into soft or hard diet groups after weaning at day 21.5. Hematoxylin-eosin and aniline blue staining were used to observe the morphology and number of odontoblasts and the amount of molar dentin formation. Immunohistochemistry was performed to observe the proliferation and apoptosis of odontoblasts. The in vivo fluorescence double-labeling was applied to evaluate the rate of molar dentin formation. RESULTS: The soft diet group had poorer periodontal membrane development but more cervical dentin deposition. Alterations in morphology and the number of odontoblasts showed a stronger correlation with age rather than food hardness. There are no significant differences in proliferative and apoptotic behavior of dentin-forming cells between the two groups. Rather, it affected the rate of dentin deposition. The rate of dentin deposition was high in the soft diet group from P21.5 to P27.5, but it was surpassed by the hard diet group within P27.5-P33.5, and the difference between the two groups disappeared at P33.5-P60.5. CONCLUSIONS: A soft diet promotes molar early cervical dentin formation. This advantage is caused by an enhanced odontoblast secretion rate rather than affecting the morphology, number, proliferation, or apoptosis of odontoblasts.

piR-368 promotes odontoblastic differentiation of dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

PURPOSE: The important role of non-coding RNAs in odontoblastic differentiation of dental tissue-derived stem cells has been widely demonstrated; however, whether piRNA (a subclass of non-coding RNA) involved in the course of odontoblastic differentiation is not yet available. This study aimed to investigate the expression profile of piRNA during odontogenic differentiation of mDPCs and the potential molecular mechanism in vitro. MATERIALS AND METHODS: The primary mouse dental papilla cells (mDPCs) were isolated from the first molars of 1-day postnatal Kunming mice. Then, they were cultured in odontogenic medium for 9 days. The expression profile of piRNA was detected by Small RNA sequencing. RT-qPCR was used to verify the elevation of piR-368. The mRNA and protein levels of mineralization markers were examined by qRT-PCR and Western blot analysis. Alkaline phosphatase (ALP) activity and alizarin red S staining were conducted to assess the odontoblastic differentiation ability. RESULTS: We validated piR-368 was significantly upregulated and interference with piR-368 markedly inhibited the odontogenic differentiation of mDPCs. In addition, the relationship between Smad1/5 signaling pathway and piR-368-induced odontoblastic differentiation has been discovered. Finally, we demonstrated Smurf1 as a target gene of piR-368 using dual-luciferase assays. CONCLUSION: This study was the first to illustrate the participation of piRNA in odontoblastic differentiation. We proved that piR-368 promoted odontoblastic differentiation of mouse dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

The ATF4-regulated LncRNA MALAT1 promotes odontoblastic differentiation of human dental pulp stem cells via histone demethylase JMJD3: An in vitro study.

AIM: This study aimed to investigate the upstream regulators and specific mechanisms of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in the odontoblastic differentiation of human dental pulp stem cells (hDPSCs). METHODOLOGY: Human dental pulp stem cells were isolated and cultured, followed by conducting loss- or gain-of-function experiments on ATF4 and loss experiments on MALAT1 to elucidate their respective biological functions in odontoblastic differentiation. Chromatin immunoprecipitation assays and RNA immunoprecipitation were performed to uncover the interaction between ATF4-MALAT1 and MALAT1-JMJD3, respectively. The odontoblastic differentiation was estimated by the mRNA and protein of DSPP and DMP1, as well as alkaline phosphatase staining. RESULTS: Expression of MALAT1 was upregulated in the hDPSCs cultured in an odontoblastic medium, and MALAT1 downregulation suppressed the odontoblastic differentiation of the hDPSCs. Subsequent experiments confirmed that ATF4 promoted odontoblastic differentiation and induced MALAT1 expression by binding to the MALAT1 promoter region. Further experiments revealed that nuclear MALAT1 interacted with JMJD3. MALAT1 knockdown decreased the JMJD3 protein level and demethylase activity, and it enhanced H3K27me3 occupancy of the promoter region of DSPP and DMP1, resulting in the inhibition of DSPP and DMP1 transcription. Importantly, JMJD3 overexpression significantly attenuated the inhibition of odontoblastic differentiation induced by MALAT1 knockdown. CONCLUSIONS: ATF4-regulated MALAT1 plays a positive regulatory role in odontoblastic differentiation of hDPSCs through JMJD3-mediated H3K27me3 modifications of the DSPP and DMP1 promoters.

Cpne7 deficiency induces cellular senescence and premature aging of dental pulp.

Once tooth development is complete, odontoblasts and their progenitor cells in the dental pulp play a major role in protecting tooth vitality from external stresses. Hence, understanding the homeostasis of the mature pulp populations is just as crucial as understanding that of the young, developing ones for managing age-related dentinal damage. Here, it is shown that loss of Cpne7 accelerates cellular senescence in odontoblasts due to oxidative stress and DNA damage accumulation. Thus, in Cpne7-null dental pulp, odontoblast survival is impaired, and aberrant dentin is extensively formed. Intraperitoneal or topical application of CPNE7-derived functional peptide, however, alleviates the DNA damage accumulation and rescues the pathologic dentin phenotype. Notably, a healthy dentin-pulp complex lined with metabolically active odontoblasts is observed in 23-month-old Cpne7-overexpressing transgenic mice. Furthermore, physiologic dentin was regenerated in artificial dentinal defects of Cpne7-overexpressing transgenic mice. Taken together, Cpne7 is indispensable for the maintenance and homeostasis of odontoblasts, while promoting odontoblastic differentiation of the progenitor cells. This research thereby introduces its potential in oral disease-targeted applications, especially age-related dental diseases involving dentinal loss.

PER2 regulates odontoblastic differentiation of dental papilla cells in vitro via intracellular ATP content and reactive oxygen species levels.

Background: Dental papilla cells (DPCs) are one of the key stem cells for tooth development, eventually forming dentin and pulp. Previous studies have reported that PER2 is expressed in a 24-hour oscillatory pattern in DPCs in vitro. In vivo, PER2 is highly expressed in odontoblasts (which are differentiated from DPCs). However, whether PER2 modulates the odontogenic differentiation of DPCs is uncertain. This research was to identify the function of PER2 in the odontogenic differentiation of DPCs and preliminarily explore its mechanisms. Methods: We monitored the expression of PER2 in DPCs differentiated in vivo. We used PER2 overexpression and knockdown studies to assess the role of PER2 in DPC differentiation and performed intracellular ATP content and reactive oxygen species (ROS) assays to further investigate the mechanism. Results: PER2 expression was considerably elevated throughout the odontoblastic differentiation of DPCs in vivo. Overexpressing Per2 boosted levels of odontogenic differentiation markers, such as dentin sialophosphoprotein (Dspp), dentin matrix protein 1 (Dmp1), and alkaline phosphatase (Alp), and enhanced mineralized nodule formation in DPCs. Conversely, the downregulation of Per2 inhibited the differentiation of DPCs. Additionally, downregulating Per2 further affected intracellular ATP content and ROS levels during DPC differentiation. Conclusion: Overall, we demonstrated that PER2 positively regulates the odontogenic differentiation of DPCs, and the mechanism may be related to mitochondrial function as shown by intracellular ATP content and ROS levels.

The impact of pediatric congenital heart disease on primary teeth structure: a histological study.

Congenital heart disease (CHD) is an abnormality in the structure or function of the cardio-circulatory system present at birth and the ventricular septal defect (VSD) is the most common CHD in children. This study aimed to determine any differences in the histological structure of primary teeth between both healthy children and those children with ventricular septal defects in Erbil City. Methods enrolled children were divided into two groups. Group I (control) & group II (CHD) aged between 6-10 years old. A total of 44 children were collected, (22 children) in each group. Enamel, dentin, and odontoblast layers were examined histologically. Unpaired t-test used for statistical analysis. Results: The histopathological sections showed a significant difference in enamel, dentin, and odontoblast layer thickness (255.8 ± 41.68- 406.4 ±46.39), (1156 ± 116.0 - 1320 ± 117.4) and (29.74 ± 7.66 -41.38 ± 12.06) respectively, with p values (P < 0.0001) for enamel and dentin layer, and P < 0.0004  for odontoblast layer. A study of the images in the CHD group showed that the tooth tissue lost its integrity and cohesion in some places, and the thickness of the enamel and dentin layer in this group was significantly reduced compared to group I.  Tissue loss in enamel, pulp, and dentin cell were observed. Also, connective tissue layers in the pulp were disrupted. Conclusions: CHD can alter the natural structure formation of primary teeth. Histologically, enamel, dentin, and odontoblasts layer thickness reduction are found in primary teeth in children with ventricular septal defects.

Fractalkine's dual role in inflammation and hard tissue formation in cultured human dental pulp cells.

This study aimed to explore the potential roles of fractalkine/CX3CR1, primarily expressed in vascular endothelial cells and has recently been identified in dental pulp cells at sites of pulp tissue inflammation, not only in inflammation but also in pulp hard tissue formation. To this end, cultured human dental pulp cells were grown in 10% FBS-supplemented α-MEM. Fractalkine was introduced to the culture, and COX-2 and dentin sialophosphoprotein (DSPP) expression levels were evaluated via western blotting. Real-time PCR was used to examine BMP-2 and Osterix mRNA expression. Calcified nodule formation was evaluated with Alizarin red staining. Results revealed that fractalkine increased COX-2 protein expression, calcified nodule formation, and BMP-2 and Osterix mRNA expression in a concentration- and time-dependent manner. DSPP protein expression also increased upon fractalkine addition. This effect of fractalkine on expression of DSPP protein was inhibited in the presence of the CX3CR1 inhibiter ADZ8797. In conclusion, our findings suggest a dual role for fractalkine in promoting pulp inflammation via COX-2 production and contributing to pulp hard tissue formation by stimulating the expression of hard tissue formation markers.