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.

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.

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.

AKT from dental epithelium to papilla promotes odontoblast differentiation.

Epithelial-mesenchymal interactions occur during tooth development. The dental epithelium (DE) is regarded as the signal center that regulates tooth morphology. However, the mechanism by which DE regulates the differentiation of mesenchyme-derived dental papilla (DP) into odontoblasts remains unclear. Using miniature pigs as a model, we analyzed the expression profiles of the DE and DP during odontoblast differentiation using high-throughput RNA sequencing. The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is one of the most enriched pathways in both DE and DP. The PI3K/AKT pathway was first activated in the inner enamel epithelium but not in the DP on embryonic day 50. This pathway was then activated in the odontoblast layer on embryonic day 60. We showed that AKT activation promoted odontoblast differentiation of DP cells. We further demonstrated that activation of PI3K/AKT signaling in the DE effectively increased the expression levels of AKT and dentin sialophosphoprotein in DP cells. Additionally, we found that DE cells secreted collagen type IV alpha 6 chain (COL4A6) downstream of epithelial AKT signaling to positively regulate mesenchymal AKT levels. Therefore, our data suggest that PI3K/AKT signaling from the DE to the DP promotes odontoblast differentiation via COL4A6 secretion.

Inductive effect of SORT1 on odontoblastic differentiation of human dental pulp-derived stem cells.

This study investigated the expression of sortilin 1 (SORT1) in cultured human dental pulp-derived stem cells (hDPSCs) and its role in their odontoblastic differentiation. Permanent teeth were extracted from five patients, and the dental pulp was harvested for explant culture. Fluorescence-activated cell sorting was used to analyze the outgrowth of adherent cells and cells that had migrated from the tissue margin. SORT1 expression was detected in hDPSCs simultaneously expressing the mesenchymal stem cell markers CD44 and CD90. The odontoblastic differentiation potential of SORT1-positive hDPSCs was examined via staining for alkaline phosphatase (ALP), an early odontoblastic differentiation marker. ALP staining was more intense in SORT1-positive than in SORT1-negative hDPSCs. Consistently, the expression of mRNA encoding SORT1 and p75NTR, a binding partner of SORT1, increased in SORT1-positive hDPSCs during odontoblastic differentiation. In addition, pro-nerve growth factor (NGF), a ligand for SORT1-p75NTR co-receptor, promoted ALP expression in SORT1-positive hDPSCs, and the interaction between SORT1 and p75NTR was detected using a coimmunoprecipitation assay. The function of SORT1 in odontoblastic differentiation was examined via RNA interference using shRNA targeting SORT1. ALP staining intensity in SORT1/shRNA-transfected cells was markedly lower than in control/shRNA-transfected cells. SORT1 knockdown decreased JUN phosphorylation and recruitment of phosphorylated JUN to the ALP promoter. Collectively, these results indicate that SORT1 is involved in the odontoblastic differentiation of hDPSCs through the JUN N-terminal kinases (JNK)/JUN signaling pathway and that the binding of SORT1 and p75NTR plays an important role in this process.

PER2 Promotes Odontoblastic/Osteogenic Differentiation of Dental Pulp Stem Cells by Modulating Mitochondrial Metabolism.

Human dental pulp stem cells (hDPSCs) possess remarkable self-renewal and multilineage differentiation ability. PER2, an essential circadian molecule, regulates various physiological processes. Evidence suggests that circadian rhythm and PER2 participate in physiological functions of DPSCs. However, the influence of PER2 on DPSCs' differentiation remains largely unknown. This study aimed to explore the effect and potential mechanism of PER2 on hDPSCs' differentiation. Dental pulp tissues were extracted, and hDPSCs were cultured for in vitro and in vivo experiments. Dorsal subcutaneous transplantation was performed in 6-week-old male BALB/c mice. The hDPSCs' odontoblastic/osteogenic differentiation was assessed, and mitochondrial metabolism was evaluated. The results indicated PER2 expression increasing during hDPSCs' odontoblastic/osteogenic differentiation. Gain- and loss-of function studies confirmed that PER2 promoted alkaline phosphatase (ALP) activity, mineralized nodules deposition, mRNA expression of DSPP, DMP1, COL1A1 and protein expression of DSPP and DMP1 in hDPSCs. Furthermore, PER2 enhanced collagen deposition, osteodentine-like tissue formation and DSPP expression in vivo. Mitochondrial metabolic evaluation aimed to investigate the mechanism of PER2-mediated hDPSC odontoblastic/osteogenic differentiation, which showed that PER2 increased ATP synthesis, elevated mitochondrial membrane potential and changed expression of proteins regulating mitochondrial dynamics. This study demonstrated that PER2 promoted hDPSCs' odontoblastic/osteogenic differentiation, which involved mitochondrial metabolic change.

Fam20C overexpression in odontoblasts regulates dentin formation and odontoblast differentiation.

FAM20C phosphorylates secretory proteins at S-x-E/pS motifs, and previous studies of Fam20C-dificient mice revealed that FAM20C played essential roles in bone and tooth formation. Inactivation of FAM20C in mice led to hypophosphatemia that masks direct effect of FAM20C in these tissues, and consequently the direct role of FAM20C remains unknown. Our previous study reported that osteoblast/odontoblast-specific Fam20C transgenic (Fam20C-Tg) mice had normal serum phosphate levels and that osteoblastic FAM20C-mediated phosphorylation regulated bone formation and resorption. Here, we investigated the direct role of FAM20C in dentin using Fam20C-Tg mice. The tooth of Fam20C-Tg mice contained numerous highly phosphorylated proteins, including SIBLINGs, compared to that of wild-type mice. In Fam20C-Tg mice, coronal dentin volume decreased and mineral density unchanged at early age, while the volume unchanged and the mineral density elevated at maturity. In these mice, radicular dentin volume and mineral density decreased at all ages, and histologically, the radicular dentin had wider predentin and abnormal apical-side dentin with embedded cells and argyrophilic canaliculi. Immunohistochemical analyses revealed that abnormal apical-side dentin had bone and dentin matrix properties accompanied with osteoblast-lineage cells. Further, in Fam20C-Tg mice, DSPP content which is important for dentin formation, was reduced in dentin, especially radicular dentin, which might lead to defects mainly in radicular dentin. Renal subcapsular transplantations of tooth germ revealed that newly formed radicular dentin replicated apical abnormal dentin of Fam20C-Tg mice, corroborating that FAM20C overexpression indeed caused the abnormal dentin. Our findings indicate that odontoblastic FAM20C-mediated phosphorylation in the tooth regulates dentin formation and odontoblast differentiation.

Effects of Red LED Irradiation in Enhancing the Mineralization of Human Dental Pulp Cells In Vitro.

Dentin regeneration is the preferred method used to preserve dental pulp vitality after pulp exposure due to caries. Red light-emitting diode irradiation (LEDI), which is based on photobiomodulation (PBM), has been used to promote hard-tissue regeneration. However, the underlying mechanism still needs elucidation. This study aimed to explore the mechanism involved in red LEDI affecting dentin regeneration. Alizarin red S (ARS) staining revealed that red LEDI induced mineralization of human dental pulp cells (HDPCs) in vitro. We further distinguished the cell proliferation (0-6 d), differentiation (6-12 d), and mineralization (12-18 d) of HDPCs in vitro and treated cells either with or without red LEDI in each stage. The results showed that red LEDI treatment in the mineralization stage, but not the proliferation or differentiation stages, increased mineralized nodule formation around HDPCs. Western blot also indicated that red LEDI treatment in the mineralization stage, but not the proliferation or differentiation stages, upregulated the expression of dentin matrix marker proteins (dentin sialophosphoprotein, DSPP; dentin matrix protein 1, DMP1; osteopontin, OPN) and an intracellular secretory vesicle marker protein (lysosomal-associated membrane protein 1, LAMP1). Therefore, the red LEDI might enhance the matrix vesicle secretion of HDPCs. On the molecular level, red LEDI enhanced mineralization by activating the mitogen-activated protein kinase (MAPK) signaling pathways (ERK and P38). ERK and P38 inhibition reduced mineralized nodule formation and the expression of relevant marker proteins. In summary, red LEDI enhanced the mineralization of HDPCs by functioning to produce a positive effect in the mineralization stage in vitro.

Dopamine is involved in reparative dentin formation through odontoblastic differentiation of dental pulp stem cells.

Conventional direct pulp-capping materials induce pulp cells to secrete various biomolecules in pulp tissues that promote reparative dentin formation through induction of odontoblastic differentiation of dental pulp stem cells (DPSCs). However, these biomolecules sometimes induce bone-like dentin with poor sealing properties. Therefore, exploration of biomolecules that allow tight sealing by tubular reparative dentin is required. We recently reported that dopamine (DA) is involved in dentinogenesis. Hence, we investigated the effect of DA on odontoblastic differentiation of DPSCs and reparative dentin formation. Both tyrosine hydroxylase (TH), a DA synthetase, and DA were expressed in odontoblast-like cells in vivo. In vitro, their expression was increased during odontoblastic differentiation of DPSCs. Furthermore, TH-overexpressing DPSCs had promoted odontoblastic differentiation and DA production. Moreover, DA stimulation promoted their differentiation and induced tubular reparative dentin. These results suggest that DA produced by TH is involved in odontoblastic differentiation of DPSCs and has an inductive capacity for reparative dentin formation similar to primary dentin. This study may lead to the development of therapy to preserve vital pulp tissues.

FGF4 and FGF9 have synergistic effects on odontoblast differentiation.

The purpose of this study was to investigate whether fibroblast growth factor 4 (FGF4) and FGF9 are active in dentin differentiation. Dentin matrix protein 1 (Dmp1) -2A-Cre transgenic mice, which express the Cre-recombinase in Dmp1-expressing cells, were crossed with CAG-tdTomato mice as reporter mouse. The cell proliferation and tdTomato expressions were observed. The mesenchymal cell separated from neonatal molar tooth germ were cultured with or without FGF4, FGF9, and with or without their inhibitors ferulic acid and infigratinib (BGJ398) for 21 days. Their phenotypes were evaluated by cell count, flow cytometry, and real-time PCR. Immunohistochemistry for FGFR1, 2, and 3 expression and the expression of DMP1 were performed. FGF4 treatment of mesenchymal cells obtained promoted the expression of all odontoblast markers. FGF9 failed to enhance dentin sialophosphoprotein (Dspp) expression levels. Runt-related transcription factor 2 (Runx2) was upregulated until day 14 but was downregulated on day 21. Compared to Dmp1-negative cells, Dmp1-positive cells expressed higher levels of all odontoblast markers, except for Runx2. Simultaneous treatment with FGF4 and FGF9 had a synergistic effect on odontoblast differentiation, suggesting that they may play a role in odontoblast maturation.

Immunohistochemical Analysis of S100 Proteins in Normal and Irreversibly Inflamed Human Dental Pulps.

INTRODUCTION: S100 proteins convey important roles in innate immune responses to infection and regenerative processes. However, their role in inflammatory or regenerative processes of the human dental pulp is poorly elucidated. The aim of the present study was to detect, localize, and compare the occurrence of 8 S100 proteins in normal, symptomatic, and asymptomatic irreversibly inflamed dental pulp specimens. METHODS: Human dental pulp specimens from 45 individuals were clinically assigned to 3 groups of pulpal diagnosis: normal pulp (NP, n = 17), asymptomatic irreversible pulpitis (AIP, n = 13), and symptomatic irreversible pulpitis (SIP, n = 15). The specimens were prepared and immunohistochemically stained for proteins S100A1, -A2, -A3, -A4, -A6, -A7, -A8, and -A9. Staining was classified using semiquantitative analysis and a 4-degree staining score (ie, no, decent, medium, and intense staining) at 4 different anatomic or functional regions (ie, the odontoblast layer [OL], pulpal stroma [PS], border area of calcifications [BAC], and vessel walls). The distribution of staining degrees between the 3 diagnostic groups was calculated using the Fisher exact text (P ≤ .05) at the 4 regions. RESULTS: Significant differences in staining were observed mainly in the OL and PS and at the BAC. The most significant differences were detected in the PS and when comparing NP with 1 of the 2 irreversibly inflamed pulpal tissues (AIP or SIP). The inflamed tissues were then invariably stained more intensely than their normal counterparts at this location (S100A1, -A2, -A3, -A4, -A8, and -A9). In the OL, NP tissue was significantly stronger stained for S100A1, -A6, -A8, and -A9 compared with SIP and for S100A9 when compared with AIP. Differences between AIP and SIP in direct comparison were rare and were found only for 1 protein (S100A2) at the BAC. Also, at the vessel walls, only 1 statistical difference in staining was observed (SIP was stronger stained than NP for protein S100A3). CONCLUSIONS: The occurrence of proteins S100A1, -A2, -A3, -A4, -A6, -A8, and -A9 is significantly altered in irreversibly inflamed compared with normal dental pulp tissue at different anatomic localizations. Some members of S100 proteins obviously participate in focal calcification processes and pulp stone formation of the dental pulp.

Identification of GPI-anchored protein LYPD1 as an essential factor for odontoblast differentiation in tooth development.

Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. These lipid raft components are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports have been published on the specific effects of lipid rafts on tooth development. Using microarray and single-cell RNA sequencing methods, we found that a GPI-AP, lymphocyte antigen-6/Plaur domain-containing 1 (Lypd1), was specifically expressed in preodontoblasts. Depletion of Lypd1 in tooth germ using an ex vivo organ culture system and in mouse dental pulp (mDP) cells resulted in the inhibition of odontoblast differentiation. Activation of bone morphogenetic protein (BMP) signaling by BMP2 treatment in mDP cells promoted odontoblast differentiation via phosphorylation of Smad1/5/8, while this BMP2-mediated odontoblast differentiation was inhibited by depletion of Lypd1. Furthermore, we created a deletion construct of the C terminus containing the omega site in LYPD1; this site is necessary for localizing GPI-APs to the plasma membrane and lipid rafts. We identified that this site is essential for odontoblast differentiation and morphological change of mDP cells. These findings demonstrated that LYPD1 is a novel marker of preodontoblasts in the developing tooth; in addition, they suggest that LYPD1 is important for tooth development and that it plays a pivotal role in odontoblast differentiation by regulating Smad1/5/8 phosphorylation through its effect as a GPI-AP in lipid rafts.

Melatonin-mediated malic enzyme 2 orchestrates mitochondrial fusion and respiratory functions to promote odontoblastic differentiation during tooth development.

Tooth development is a complex process that is tightly controlled by circadian rhythm. Melatonin (MT) is a major hormonal regulator of the circadian rhythm, and influences dentin formation and odontoblastic differentiation during tooth development; however, the underlying mechanism remains elusive. This study investigated how MT regulates odontoblastic differentiation, with a special focus on its regulation of mitochondrial dynamics. In rat dental papilla cells (DPCs), we found that MT promotes odontoblastic differentiation concurrently with enhanced mitochondrial fusion, while disruption of mitochondrial fusion by depleting optic atrophy 1 (OPA1) impairs MT-mediated differentiation and mitochondrial respiratory functions. Through RNA sequencing, we discovered that MT significantly upregulated malic enzyme 2 (ME2), a mitochondrial NAD(P)+ -dependent enzyme, and identified ME2 as a critical MT downstream effector that orchestrates odontoblastic differentiation, mitochondrial fusion, and respiration functions. By detecting the spatiotemporal expression of ME2 in developing tooth germs, and using tooth germ reconstituted organoids, we also provided in vivo and ex vivo evidence that ME2 promotes dentin formation, indicating a possible involvement of ME2 in MT-modulated tooth development. Collectively, our findings offer novel understandings regarding the molecular mechanism by which MT affects cell differentiation and organogenesis, meanwhile, the critical role of ME2 in MT-regulated mitochondrial functions is also highlighted.

Phosphorylation of ATF2 promotes odontoblastic differentiation via intrinsic HAT activity.

Mouse dental papilla cells (mDPCs) are cranial neural crest-derived dental mesenchymal cells that give rise to dentin-secreting odontoblasts after the bell stage during odontogenesis. The odontoblastic differentiation of mDPCs is spatiotemporally regulated by transcription factors (TFs). Our previous work reveals that chromatin accessibility was correlated with the occupation of the basic leucine zipper TF family during odontoblastic differentiation. However, the detailed mechanism by which TFs regulate the initiation of odontoblastic differentiation remains elusive. Here, we report that phosphorylation of ATF2 (p-ATF2) is particularly increased during odontoblastic differentiation in vivo and in vitro. ATAC-seq and p-ATF2 CUT&Tag experiments further demonstrate a high correlation between p-ATF2 localization and increased chromatin accessibility of regions near mineralization-related genes. Knockdown of Atf2 inhibits the odontoblastic differentiation of mDPCs, while overexpression of p-ATF2 promotes odontoblastic differentiation. ATAC-seq after overexpression of p-ATF2 reveals that p-ATF2 increases the chromatin accessibility of regions adjacent to genes associated with matrix mineralization. Furthermore, we find that p-ATF2 physically interacts with and promotes H2BK12 acetylation. Taken together, our findings reveal a mechanism that p-ATF2 promotes odontoblastic differentiation at initiation via remodeling chromatin accessibility and emphasize the role of the phosphoswitch model of TFs in cell fate transitions.

Iroquois homeobox 3 regulates odontoblast proliferation and differentiation mediated by Wnt5a expression.

Iroquois homeobox (Irx) genes are TALE-class homeobox genes that are evolutionarily conserved across species and have multiple critical cellular functions in fundamental tissue development processes. Previous studies have shown that Irxs genes are expressed during tooth development. However, the precise roles of genes in teeth remain unclear. Here, we demonstrated for the first time that Irx3 is an essential molecule for the proliferation and differentiation of odontoblasts. Using cDNA synthesized from postnatal day 1 (P1) tooth germs, we examined the expression of all Irx genes (Irx1-Irx6) by RT-PCR and found that all genes except Irx4 were expressed in the tooth tissue. Irx1-Irx3 a were expressed in the dental epithelial cell line M3H1 cells, while Irx3 and Irx5 were expressed in the dental mesenchymal cell line mDP cells. Only Irx3 was expressed in both undifferentiated cell lines. Immunostaining also revealed the presence of IRX3 in the dental epithelial cells and mesenchymal condensation. Inhibition of endogenous Irx3 by siRNA blocks the proliferation and differentiation of mDP cells. Wnt3a, Wnt5a, and Bmp4 are factors involved in odontoblast differentiation and were highly expressed in mDP cells by quantitative PCR analysis. Interestingly, the expression of Wnt5a (but not Wnt3a or Bmp4) was suppressed by Irx3 siRNA. These results suggest that Irx3 plays an essential role in part through the regulation of Wnt5a expression during odontoblast proliferation and differentiation.

SVCT2-GLUT1-mediated ascorbic acid transport pathway in rat dental pulp and its effects during wound healing.

Ascorbic acid (AA; vitamin C) plays a crucial role in the biosynthesis and secretion of collagen to produce the organic matrix of hard tissues. Nevertheless, the detailed mechanism by which AA induces reparative dentinogenesis is still unknown. This study aimed to investigate the pathway and function of AA during wound healing in a rat pulpotomy model. Sodium-dependent vitamin C transporter (SVCT) 2 and glucose transporter (GLUT) 1 were detected in odontoblasts, endothelial cells, and nerve fibers in normal pulp tissues. SVCT2 and GLUT1 were also expressed in odontoblast-like cells in pulpotomized tissues of Wistar rats, and immunopositive cells of SVCT2 were significantly increased at 5 days after pulpotomy (p < 0.05). By contrast, osteogenic disorder Shionogi (ODS) rats, which cannot generate AA, also expressed SVCT2 and GLUT1 in normal and wound healing conditions. However, in ODS rats, when compared with the AA-addition group, the formation of dentin bridges in the AA-loss group was not evident, a layer of osteopontin was significantly increased beneath the wound surface (p < 0.05), and alpha smooth muscle actin at the odontoblast-like cells observed along this layer was significantly increased (p < 0.05), but not Nestin. Moreover, the amounts of type 1 collagen generated in the reparative dentin and beneath the wound healing site were significantly diminished (p < 0.05). Macrophages expressing CD68 and CD206 increased beneath the wound site. Hence, AA may be involved in odontoblast-like cell differentiation and anti-inflammatory response during dental pulp wound healing. Our results provide new insights into the function of AA through SVCT2 and GLUT1 in reparative dentinogenesis and may help in developing new therapeutic targets for dental pulpal disease.

Platelet-rich Fibrin Promotes the Proliferation and Osteo-/odontoblastic Differentiation of Human Dental Pulp Stem Cells.

BACKGROUND: Pulp regeneration is a promising strategy that promotes the continued development of young permanent teeth with immature apical foramen. Platelet-rich fibrin (PRF) was found to stimulate the proliferation and differentiation of osteoblasts, but its effects on osteoblast/odontoblast differentiation of human dental pulp stem cells (hDPSCs) are unknown. METHODS: The hDPSCs were isolated and identified using known surface markers by flow cytometry. The CCK-8 assay and the expression of Ki67 and PCNA were used to examine hDPSC proliferation. After 7 days of culture in an osteo-/odontoblastic induction medium with various concentrations of liquid PRF (0, 10% and 20%), the early stage of osteogenesis-intracellular alkaline phosphatase (ALP) was checked. After 21 days of culture, matrix mineralization was checked using Alizarin Red S and quantified. The mRNA and protein levels of osteo-/odontoblastic genes, including RUNX2, DSPP, DMP1 and BSP, were measured by qRT-PCR. The notch signal was checked by Western blot to analyze three key proteins (Notch 1, Jagged 1 and Hes 1). RESULTS: PRF-treated groups showed higher expression of Ki-67 and PCNA, higher ALP activity, and the higher dose showed a stronger induction. PRF promoted osteo-/odontoblastic differentiation of hDPSCs indicated by elevated protein levels and mRNA levels of the expression of osteo-/odontoblastic markers. The three key proteins in Notch signaling showed an increase compared with the control group and increased as the PRF concentration increased. CONCLUSION: PRF can promote the proliferation and osteo-/odontoblastic differentiation of hDPSC, which may be through the Notch signaling pathway.

Important roles of odontoblast membrane phospholipids in early dentin mineralization.

The objective of this study was to first identify the timing and location of early mineralization of mouse first molar, and subsequently, to characterize the nucleation site for mineral formation in dentin from a materials science viewpoint and evaluate the effect of environmental cues (pH) affecting early dentin formation. Early dentin mineralization in mouse first molars began in the buccal central cusp on post-natal day 0 (P0), and was first hypothesized to involve collagen fibers. However, elemental mapping indicated the co-localization of phospholipids with collagen fibers in the early mineralization area. Co-localization of phosphatidylserine and annexin V, a functional protein that binds to plasma membrane phospholipids, indicated that phospholipids in the pre-dentin matrix were derived from the plasma membrane. A 3-dimensional in vitro biomimetic mineralization assay confirmed that phospholipids from the plasma membrane are critical factors initiating mineralization. Additionally, the direct measurement of the tooth germ pH, indicated it to be alkaline. The alkaline environment markedly enhanced the mineralization of cell membrane phospholipids. These results indicate that cell membrane phospholipids are nucleation sites for mineral formation, and could be important materials for bottom-up approaches aiming for rapid and more complex fabrication of dentin-like structures.

25-Hydroxycholesterol induces odontoclastic differentiation through RANK-RANKL upregulation and NF-κB activation in odontoblast-like MDPC-23 cells: An in vitro study.

AIM: The physiological effects and cellular mechanism of 25-hydroxycholesterol (25-HC), which is an oxysterol synthesized from cholesterol by cholesterol-25-hydroxylase (CH25H) expressed under inflammatory conditions, are still largely unknown during odontoclastogenesis. This study aimed to evaluate 25-HC-induced odontoclastogenesis and its cellular mechanisms in odontoblast-like MDPC-23 cells. METHODOLOGY: To investigate 25-HC-induced odontoclastogenesis of MDPC-23 cells and its cellular mechanism, haemotoxylin and eosin staining, tartrate-resistant acid phosphatase (TRAP) staining, dentine resorption assay, zymography, reactive oxygen species (ROS) detection, immunocytochemistry, and nuclear translocation were performed. The experimental values are presented as mean ± standard deviation and were compared using analysis of variance, followed by post hoc multiple comparisons (Tukey's test) using SPSS software version 22 (IBM Corp.). A p-value <.05 was considered statistically significant. RESULTS: Lipopolysaccharide or receptor activator of nuclear factor-κB ligand (RANKL) induced the synthesis of 25-HC via the expression of CH25H in MDPC-23 cells (p < .01). Multinucleated giant cells with morphological characteristics and TRAP activity of the odontoclast were increased by 25-HC in MDPC-23 cells (p < .01). Moreover, 25-HC increased dentine resorption through the expression and activity of matrix metalloproteinases in MDPC-23 cells. It not only increased the expression of odontoclastogenic biomarkers but also translocated cytosolic nuclear factor-κB (NF-κB) to the nucleus in MDPC-23 cells. Additionally, 25-HC not only increased the production of ROS (p < .01), expression of inflammatory mediators (p < .01), pro-inflammatory cytokines, receptor activator of NF-κB (RANK), and RANKL but also suppressed the expression of osteoprotegerin (OPG) in MDPC-23 cells. In contrast, CDDO-Me, a chemical NF-κB inhibitor, decreased TRAP activity (p < .01) and downregulated the expression of the odontoclastogenic biomarkers, including RANK and RANKL, in MDPC-23 cells. CONCLUSION: 25-HC induced odontoclastogenesis by modulating the RANK-RANKL-OPG axis via NF-κB activation in MDPC-23 cells. Therefore, these findings provide that 25-HC derived from cholesterol metabolism may be involved in the pathophysiological etiological factors of internal tooth resorption.