The odontoblastic differentiation of dental mesenchymal stem cells: molecular regulation mechanism and related genetic syndromes

Dental mesenchymal stem cells (DMSCs) are multipotent progenitor cells that can differentiate into multiple lineages including odontoblasts, osteoblasts, chondrocytes, neural cells, myocytes, cardiomyocytes, adipocytes, endothelial cells, melanocytes, and hepatocytes. Odontoblastic differentiation of DMSCs is pivotal in dentinogenesis, a delicate and dynamic process regulated at the molecular level by signaling pathways, transcription factors, and posttranscriptional and epigenetic regulation. Mutations or dysregulation of related genes may contribute to genetic diseases with dentin defects caused by impaired odontoblastic differentiation, including tricho-dento-osseous (TDO) syndrome, X-linked hypophosphatemic rickets (XLH), Raine syndrome (RS), hypophosphatasia (HPP), Schimke immuno-osseous dysplasia (SIOD), and Elsahy-Waters syndrome (EWS). Herein, recent progress in the molecular regulation of the odontoblastic differentiation of DMSCs is summarized. In addition, genetic syndromes associated with disorders of odontoblastic differentiation of DMSCs are discussed. An improved understanding of the molecular regulation and related genetic syndromes may help clinicians better understand the etiology and pathogenesis of dentin lesions in systematic diseases and identify novel treatment targets.


Introduction
Dentin is a thick and highly mineralized tissue layer under the enamel that protects the dental pulp cavity from infections, supports and provides nutrition to the enamel, and alleviates dental pressure (Lopez-Cazaux et al., 2006;Liu et al., 2022b).They are formed from odontoblasts (Zhang et al., 2005;Martens et al., 2013).Dentin formation, also known as dentinogenesis, begins with the differentiation of odontoblasts.Odontoblasts are derived from the neural crest-derived mesenchymal cells (Cobourne and Sharpe, 2003).Odontoblasts first occur at the principal cusp tip and then proceed to the base of the tooth, suggesting a spatiotemporal pattern of odontoblast differentiation (Thesleff, 2003;Chen et al., 2008).Odontoblastic differentiation is regulated by a network encompassing signaling pathways, transcriptional factors (TFs), and posttranscriptional and epigenetic regulation.However, any problems in the regulatory network affect dentin development, most of which appear to be genetic syndromes with dentin defects caused by impaired odontoblast differentiation.Therefore, the molecular regulatory mechanism of odontoblastic differentiation of dental mesenchymal stem cells (DMSCs) is discussed in this review, and genetic syndromes associated with odontoblastic differentiationrelated dentin lesions are also discussed.

Wnt signaling pathway
The first member of the Wnt family, the Wnt1 gene, was discovered by Nusse andVarmus in 1982 (Nusse andClevers, 2017), since then, there have been numerous studies on the Wnt pathway.The Wnt signal transduction cascade is critical for the regulation of development, control of stem cells, and disease (Nusse and Clevers, 2017).Moreover, the Wnt pathway is important for odontoblast differentiation (Chen et al., 2009a).
The canonical Wnt/β-catenin pathway plays an important role in odontoblastic differentiation.Wnt10a mediates expression of dentin sialophosphoprotein (Dspp), an upstream regulatory molecule.It is critical for dentinogenesis and odontoblastic differentiation (Yamashiro et al., 2007).Lef1 is important for odontoblast differentiation because it upregulates DSPP and osteocalcin (OCN) mRNA expression in dental pulp cells (DPCs) (Yokose and Naka, 2010).β-catenin knockdown results in decreased odontoblastic differentiation.Mechanistically, βcatenin activates runt-related transcription factor 2 (Runx2), thereby enhancing odontoblastic differentiation of DPCs during reparative dentin formation (Han et al., 2014).In vitro study has suggested that β-catenin signaling enhances the formation of preodontoblasts.The number of pre-odontoblasts and odontoblasts increased after the exposure of DPCs to Wnt3a.Expression of dentine matrix protein 1 (Dmp1) and Dspp is upregulated in DPCs exposed to Wnt3a (Vijaykumar et al., 2021).In vivo stem cell implantation assay suggested that the synergistic action of bone morphogenetic protein 9 (BMP9) and Wnt3a may enhance the odontoblastic differentiation of immortalized mouse stem cells of the apical papilla tissue of mouse lower incisor teeth (iSCAPs) (Zhang et al., 2015a).Wntless (Wls) is a Wnt chaperone protein that is essential for Wnt signaling.Deletion of the Wls gene reduces activation of the Wnt pathway and downregulates Runx2 levels, thereby disrupting the homeostasis of odontoblast differentiation (Lim et al., 2014).Odontoblast-specific deletion of the Wls gene leads to the downregulation of Wnt10a, β-catenin, collagen type I (Col1), and dentin sialoprotein (DSP), leading to reduced canonical Wnt activity and inhibition of odontoblast maturation (Bae et al., 2015).In vivo study showed that the deletion of Wls or overexpression of the Wnt antagonist Dkk1 decreased odontoblastic differentiation by inhibiting Wnt signaling (Zhang et al., 2021b).These results demonstrate the involvement of β-catenin pathway in odontoblastic differentiation.

FIGURE 1
Isolation and differentiation of dental mesenchymal stem cells (DMSCs).Dental pulp stem cells (DPSCs) are located in dental pulp of permanent teeth, stem cells from human exfoliated deciduous teeth (SHEDs) are located in immature dental pulp of deciduous teeth, and stem cells from apical papilla (SCAPs) are located in root apical papilla tissue on the exterior of the root foramen area.These three DMSCs can be isolated by enzymatic solution and explant method.DPSCs, SHEDs, and SCAPs can be cultured in the same induction medium of L-ascorbate-2-phosphate, dexamethasone, and inorganic phosphate to undergo the odontoblastic differentiation process.While under appropriate induction conditions, they can differentiate into a variety of cells besides odontoblasts.For example, DPSCs can differentiate into odontoblasts, osteoblasts, adipocytes, neural cells, cardiomyocytes, myocytes, chondrocytes, melanocytes, and hepatocytes.SHEDs can differentiate into osteogenic, chondrogenic, adipogenic cells, neural cells, odontoblasts, endothelial cells, and hepatocytes.And SCAPs can differentiate into adipocytes, odontoblasts, and osteoblasts.(Zhang et al., 2019).Matrix-metalloproteinase-13 (MMP-13) interacts with Wnt/β-catenin pathway, as in MMP13knockout mice, the Wnt-responsive gene Axin2 was downregulated and dentin formation was defected (Duncan et al., 2022).Herbal extracts also promote odontoblast differentiation of DPSCs via Wnt/ β-catenin pathway, such as Berberine, Baicalein, and Wedelolactone (Lee et al., 2016;Wang et al., 2018;Wu et al., 2019).Exosome-like vesicles derived from the Hertwig's epithelial root sheath (HERS) cell line (ELVs-H1) boosts the migration and proliferation of DPCs.And ELVs-H1 also promotes odontogenic differentiation through activation of the Wnt/β-catenin pathway (Zhang et al., 2020b).Suppression of Wnt/β-catenin pathway is related to the inhibition of odontoblastic differentiation.Knockdown of special AT-rich sequence-binding protein 2 (SATB2) leads to decreased βcatenin levels and increased DKK1 expression, resulting in the inhibition of odontoblastic differentiation of hDPSCs (Xin et al., 2021).Lead (Pb) inhibits Wnt/β-catenin pathway and thus impairs odontoblastic differentiation of hDPSC (Khalid et al., 2022).Nevertheless, there is still conflict regarding the role of Wnt/βcatenin signaling in odontoblast differentiation.Odontoblastic differentiation of hDPSCs can be enhanced by the long noncoding RNA (lncRNA) short nucleolar RNA host gene 1 (SNHG1), in which Wnt/β-catenin pathway is inhibited by microRNA-328-3p (miR-328-3p) (Fu et al., 2022).
However, Smad3, a regulator of TGF-β signaling, may play a negative regulatory role in odontoblastic differentiation.In MDPC-23 cells, the overexpression of Smad3 diminishes DSPP gene transcription by increasing the inhibitory ability of TGF-β1 (He et al., 2004).In contrast, ALP and OCN mRNA expression is upregulated in SCAPs treated with TGF-β1 and Smad3 inhibitors (He et al., 2014).The knockdown of Smad3 promotes the odontoblastic differentiation of DPSCs.As observed in  mineralization-induced DPSCs, the knockdown of Smad3 induces the early expression of DSPP and DMP1, and ALP expression is increased (Huang et al., 2019).

TGF-β superfamily/non-Smad signaling pathway
Besides, non-Smad signaling pathways are also activated by TGF-β superfamily members in the process of odontoblastic differentiation.In hDPCs, BMP2 activates p38a MAPK as it induces the phosphorylation of p38α depending on dose and time, and p38 MAPK pathway regulates the stimulation of BMP2 (Qin et al., 2012a;Yang et al., 2015).Qin et al. first demonstrated that in BMP2-induced DPCs, JNK MAPK is specifically implicated in the late-stage of odontoblastic differentiation (Qin et al., 2014).While in the early phase of odontoblastic differentiation in DPSCs, TGF-β1 enhances odontoblastic differentiation via AKT, Erk1/2 and p38 MAPK signaling pathways, instead of Smad3 or JNK pathways (Bai et al., 2023) (Figure 3).

Mitogen-activated protein kinase (MAPK) signaling pathway
The MAPK pathway controls various physiological processes, including cell proliferation, gene expression, and apoptosis.This is mediated by ERK, c-Jun amino-terminal kinase (JNK), and p38 protein kinases (Johnson and Lapadat, 2002).ERK controls mitosis, JNK regulates transcription and inflammatory cytokines, and environmental stress may activate p38 (Johnson and Lapadat, 2002).MAPK pathways can be divided into conventional and atypical pathways.The conventional MAPK pathway consists of the classical cascade of MAP kinase kinase kinase (MAPKKK), MAP kinase kinase (MAPKK or MEK), and MAPK, in which MAPK is the effector-phosphorylating substrate.Typical MAPKs, including p38s, ERK1/ERK2, JNKs, and ERK5, possess a distinct Thr-Xaa-Tyr motif in the activation loop and thus can be activated by MAPKKs.However, atypical MAPK pathway lacks the three-tiered MAPKKK-MAPKK-MAPK cascade, and related MAPKs are absent of the Thr-Xaa-Tyr motif, such as ERK3/4, ERK7, and Nemo-like kinase (NLK) (Coulombe and Meloche, 2007).In this review, we summarize the roles of the three best-known conventional MAPK pathways in odontoblastic differentiation (Figure 4).
The ERK pathway is critical for the promotion of odontoblastic differentiation.The MEK/ERK pathway contributes to the activation and phosphorylation of RUNX2 (Xiao et al., 2002), a pivotal transcription factor (TF) related in odontoblastic differentiation.When the ERK signaling pathway was blocked by the specific inhibitor U0126, the levels of odontoblastic markers were significantly downregulated (Karanxha et al., 2013).Moreover, it is associated with various factors involved in odontogenesis regulation as shown in Figure 2 and Table 1.For instance, in hDPCs, the ERK pathway functions positively during simvastatin-induced odontoblastic differentiation (Karanxha et al., 2013).Mechanical stress promotes odontoblastic differentiation of SCAPs through the ERK and JNK pathways, where the protein levels of pERK and pJNK are upregulated (Mu et al., 2014).Calcium hydroxide (Ca(OH) 2 ) enhances DPSC differentiation by promoting the expression of p38, JNK, and ERK (Chen et al., 2016b).EREG also enhances ERK1/ 2 phosphorylation to increase odontoblastic differentiation of DPSCs (Cui et al., 2019).N-acetylneuraminic acid (Neu5Ac) promotes odontoblastic differentiation of DPSCs by activating the ERK pathway (Li et al., 2021).However, odontoblastic differentiation of hDPSCs is diminished by delta-like homolog 1 (DLK1) via the ERK pathway (Qi et al., 2017).
The JNK pathway has also been implicated in odontoblast differentiation.In hDPCs, Wnt6 enhances the JNK signaling pathway, thus increasing cell migration and differentiation; however, inhibition of the JNK pathway reduces Wnt6-induced odontoblastic differentiation (Li et al., 2014).In contrast, the suppression of JNK MAPK attenuates the odontoblastic differentiation of DPCs in the late phase (Qin et al., 2014) (Figure 3).

Neurogenic locus notch homolog (Notch) signaling pathway
Notch signaling is involved in cell differentiation, proliferation, and death (Cai et al., 2011).Four Notch pathway receptors have been identified: Notch1, Notch2, Notch3, and Notch4.Notch ligands are divided into two categories: typical Notch ligand, including DLL-type ligands (Delta-like1, Delta-like3 and Delta-like4) and JAG-type ligands (Jagged1, Jagged2), and atypical Notch ligands, including DNER, F3⁄ Contactin, and NB-3 (Eiraku et al., 2002;Cai et al., 2011).Compared to atypical ligands, typical ligands have a conserved DSL domain and higher affinity (Cai et al., 2011).When Notch ligands bind to their receptors, metalloprotease TNF-a converting enzyme and c-Secretase complex may cut the S2 and S3 sites of the receptors, and the Notch intracellular domain (NICD) is formed.NICD transmits signals to the nucleus and induces gene transcription (Cai et al., 2011).Notch signaling consists of the canonical and non-canonical Notch pathways.The canonical Notch pathway can be briefly identified as an NICD-CSL-MAML cascade activated by interactions between typical Notch ligands and receptors.Noncanonical Notch pathway is not well known, but it has different target genes and mediators than the canonical pathways (Cai et al., 2011).
The Notch signaling pathway is also involved in odontoblastic differentiation (Figure 4).Notch receptors and ligands are found in the dental epithelium or mesenchyme during odontogenesis, suggesting that Notch signaling may be involved in dentin formation (Zhang et al., 2008).In vitro study demonstrates that Notch2-Delta signaling positively mediates the odontoblastic differentiation of DPCs (He et al., 2003).The Notch pathway is activated by various stimulants, as shown in Figure 2 and Table 1.In vivo study showed that the Notch pathway was activated after pulp capping with Ca(OH) 2 , as increased expression of Notch1, Notch2, Notch3, Delta1, Jagged1 and Hes1 was observed.This suggests that pulp capping with Ca(OH) 2 promotes odontoblastic differentiation of DPCs (Løvschall et al., 2005).Notch1, Notch2, ALP, OPN, and OCN mRNAs are upregulated in rat DPCs treated with tenascin-C (TN-C), suggesting that TN-C induces odontogenic differentiation through the Notch pathway (Matsuoka et al., 2013).Human βdefensin 4 (HBD4) increases odontoblast differentiation of SHEDs and DPSCs through the Notch pathway; therefore, HBD4 may be a prospective agent for pulp capping (Zhai et al., 2019;Zhai et al., 2020).Platelet-rich fibrin (PRF) increases the expression of important Notch signaling proteins such as Notch1, Jagged1, and Hes1, and upregulates odontoblastic markers in hDPSCs.Thus, PRF enhances odontoblastic differentiation (Zhang et al., 2022).Overexpression of periostin (Postn) inhibited odontoblast differentiation of mDPCs, and downregulation of Notch signaling molecules was observed.Therefore, downregulation of the Notch pathway adversely affects odontoblastic differentiation (Zhou et al., 2015).These results demonstrate the positive role of the Notch pathway.
However, Notch signaling can negatively affect odontoblastic differentiation.Zhang et al. first demonstrated that overexpressed Jagged1 activates the Notch pathway; consequently, odontoblastic differentiation of DPSCs is decreased in vitro and in vivo (Zhang et al., 2008).Overexpression of NICD diminishes the odontoblastic differentiation of DPSCs, suggesting a negative effect on the Notch pathway.DSPP expression is downregulated by Notch signaling, and mechanistically, Notch signaling may inhibit Runx2-dependent gene transcription via the Notch target gene Hes1 (Zhang et al., 2008).Moreover, hDPSCs with inhibition of Delta1 tend to differentiate into odontoblasts compared with the control group (Wang et al., 2011).

Transcriptional factors (TFs)
TFs are important for many physiological processes.Different signaling pathways are linked by TFs, which subsequently initiate specific gene expression by binding to enhancers and promoters (Tao et al., 2019).Recently, TFs were reported to be involved in dentinogenesis.Frequently mentioned odontoblast-related TFs are summarized in this review, including RUNX2, SP7, DLX3, KLF4, NFIC etc. (Figure 4).

Homeobox gene distal-less 3 (DLX3)
DLX3 is a member of the DLX family and is involved in tooth development (Ghoul-Mazgar et al., 2005).During odontoblast differentiation, Dlx3 expression increases at mRNA and protein levels (Li et al., 2012).In hDPCs, Dlx3 upregulated ALP activity and DSPP and DMP1 levels.Therefore, Dlx3 enhances odontoblastic differentiation of hDPCs (Li et al., 2012).In a mouse model knockout of Dlx3 in the neural crest, defective odontoblast differentiation and impaired dentin formation were observed, and DSPP levels decreased significantly (Duverger et al., 2012).Dlx3 directly regulates Oc and Runx2 in ex vivo studies on bone (Duverger et al., 2012), and Runx2 is a critical TF in odontoblastic differentiation.

Krüppel-like factor 4 (KLF4)
KLF4 is homogenic to the Drosophila melanogaster Krüppel protein and is pivotal during odontoblastic differentiation (Tao et al., 2019).Expression of KLF4 been observed in the mouse polarizing odontoblast layer, suggesting that KLF4 may positively affect odontoblastic differentiation at the terminal stage (Chen et al., 2009b).In hDPCs, overexpression of KLF4 results in increased levels of ALP and the odontogenic markers DMP1 and DSPP (Lin et al., 2011a).
Mechanistically, KLF4 promotes odontoblastic differentiation by activating TGF-β signaling pathway in the initial stage, in which Runx2 is a cofactor.KLF4 also increases the transcription of Dmp1 and Sp7 by binding to their promoters and regulating histone acetylation.KLF4 interacts with HDAC3 and P300 during the early and later stages of odontoblastic differentiation, respectively (Lin et al., 2013;Tao et al., 2019).Enlarged pulp canals and dentin mineralization defects have been observed in Klf4-knockout mouse models (Tao et al., 2019).When Klf4 transcription is activated by the binding of NFIC through binding to its promoter, the expression of Dmp1 and DSPP is elevated, thus promoting odontoblast differentiation (Lee et al., 2014).In conclusion, KLF4 promotes odontoblastic differentiation.

Others
In addition to the TFs mentioned above, many other TFs participate in the regulation of odontoblastic differentiation in DMSCs.In the mouse immortalized dental papilla mesenchymal cell line (iMDP-3), overexpression of Klf5, Klf6, and Klf10 promotes odontoblastic differentiation.Mechanistically, the transcription of Dspp and Dmp1 is enhanced by Klf5, Klf6, and Klf10 (Chen et al., 2016c;Chen et al., 2017;Chen et al., 2021).ATF6, an endoplasmic reticulum (ER) membrane-bound TF, is involved in hDPC odontoblastic differentiation.Overexpression of ATF6 results in an increased expression of DSPP and DMP1 (Kim et al., 2014).The expression of zinc finger E-box-binding homeobox 1 (Zeb1) is observed in the tooth germ mesenchyme and increases during odontogenic differentiation in vivo and in vitro.When Zeb1 is inhibited, the differentiation of mDPCs is therefore reduced.Mechanistically, the expression of Runx2 and Dspp is promoted by ZEB1 respectively in the early and late phases of odontoblastic differentiation (Xiao et al., 2021).BTB and CNC homology 1 (BACH1) is a transcription repressor present in the odontoblast layer.Upregulation and downregulation of BACH1 induces odontoblastic differentiation in a positive and negative manner, respectively, by interacting with HO-1 (Liu et al., 2022a).Hypoxiainducible factor 1 (HIF1) is a TF activated by hypoxic circumstances, whose subunit HIF1α proves to enhance odontoblast differentiation.In SHEDs from patients with fibrodysplasia ossificans progressiva, BMP pathway is activated by HIF1α, thus odontoblast differentiation is promoted (Wang et al., 2016).Moreover, HIF1α upregulates odontogenic differentiation of hDPSCs via Wnt/βcatenin pathway synergically with BCL9 (Orikasa et al., 2022).

Posttranscriptional regulation
In the process of odontoblastic differentiation, posttranscriptional regulation is involved in odontoblastic differentiation.This review provides an overview of the regulation by microRNAs (miRNAs, miRs) and long noncoding RNAs (lncRNAs) at the posttranscriptional level.
H19 promotes odontoblastic differentiation at the posttranscriptional level.During odontoblastic differentiation of hDPSCs, notably increased expression of H19 has been observed (Zhong et al., 2020).In vitro and in vivo studies suggest that odontoblastic differentiation of hDPSCs and SCAPs can be enhanced by the overexpression of H19 but inhibited by its downregulation (Li et al., 2019;Zhong et al., 2020).H19 promotes the odontoblastic differentiation of hDPSCs via the H19/SAHH axis (Zeng et al., 2018a).The odontoblastic differentiation of SCAPs is promoted by H19 via the miR-141/ SPAG9 pathway (Li et al., 2019).Mechanistically, H19 interacts with miRNAs.For instance, H19 stops the miRNA-mediated degradation of SPAG9 by competitively binding to miR-141; therefore, the phosphorylation of p38 and JNK is increased and the differentiation of SCAPs is promoted (Li et al., 2019).Moreover, H19 attenuates miR-140-5p′s inhibitory activity on odontoblastic differentiation by acting as a sponge for miR-140-5p.Therefore, H19 promotes the expression of BMP2 and FGF9, and enhances hDPSCs differentiation into odontoblasts (Zhong et al., 2020).These results demonstrated the positive function of H19 in the regulation of odontoblastic differentiation.
However, the lncRNA DANCR has a negative impact on odontoblastic differentiation.Kretz et al. first identified an lncRNA and named it anti-differentiation non-coding RNA (ANCR), and subsequently named it DANCR (Kretz et al., 2012).The expression of DANCR decreases during odontoblastic differentiation of hDPCs in a time-dependent manner (Chen et al., 2016a), indicating that DANCR may be negative for hDPCs differentiation.In hDPCs overexpressing DANCR, the levels of odontogenic markers, such as DSPP and DMP1, are downregulated.And upregulation of DANCR leads to lowered expression of phosphorylation of GSK3β (p-GSK3β) and β-catenin, suggesting the inhibition of Wnt/β-catenin signal pathway and odontoblastic differentiation (Chen et al., 2016a).Moreover, DANCR acts as a sponge of miR-216a by directly binding to it.Therefore, DANCR sponges miR-216a to inhibit the odontoblastic differentiation of hDPCs by enhancing the expression of c-CBL, which suppresses odontoblastic differentiation but can be inhibited by miR-216a (Chen et al., 2020).These results suggest a negative role for DANCR in odontoblastic differentiation.

Epigenetic regulation
Epigenetic regulation, including DNA methylation and histone tail modification, has recently been shown to modulate odontoblastic differentiation, as shown in Figure 4 (Kamiunten et al., 2015).

DNA methylation
DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, and DNMT3B, regulate DNA methylation, which tends to silence the promoter and enhancer classes (Smith and Meissner, 2013).DNA methylation is an important epigenetic regulator of odontoblast differentiation.The loss of ten-eleven translocation 1 (TET1), a DNA methyl cytosine dioxygenase, arrests hydroxymethylation and transcription of the Family with Sequence Similarity 20C (FAM20C), thereby inhibiting odontoblastic differentiation of hDPCs (Li et al., 2018a).In DPSCs, the H19/SAHH axis enhances odontoblastic differentiation by diminishing the methylation of DLX3 mediated by DNMT3B (Zeng et al., 2018a).In pre-odontoblastic cells, loss of DNMTs promotes odontoblastic differentiation by elevating the expression of Klf4 and odontoblastic marker genes.SP1 modulates KLF4 via a demethylated binding site on a CpG island in KLF4 promoter region (Sun et al., 2019).N6-methyladenosine (m 6 A) methyltransferase METTL3 promotes odontoblastic differentiation.Absence of METTL3 in hDPCs inhibits NFIC translation.Consequently, the knockdown of METTL3 results in decreased odontogenic differentiation in vitro, and reduced dentin formation in the root has been observed in vivo (Sheng et al., 2021).These results demonstrate the dual role of DNA methylation in odontoblastic differentiation.

Histone modification
Epigenetic modifications of histones include methylation, acetylation, phosphorylation, and ubiquitylation (Chen and Dent, 2014), among which methylation and acetylation are the most frequently mentioned histone modifications that are involved in odontoblastic differentiation.
However, HDMs and KDMs sometimes suppress odontoblastic differentiation.For instance, FBXL11 (KDM2A) binds to the BCL6 co-repressor for activation; consequently, EREG transcription is inhibited by the increased methylation of histone K4/36 in the EREG promoter.Therefore, FBXL11 inhibits odontoblastic differentiation of SCAPs (Du et al., 2013).KDM5A negatively modulates the odontoblastic differentiation of hDPCs by deleting H3K4me3 from the promoters of target genes, and the inhibition of KDM5A increases H3K4me3 levels, as well as ALP activity and odontogenic markers (Li et al., 2020).Inhibition of HDMs leads to the upregulation of H3K4me3 and promotes odontoblastic differentiation (Yuan et al., 2022).

Histone acetylation
Histone acetyltransferases (HATs) and deacetylases (HDACs) are responsible for histone acetylation, which changes the interaction between histone proteins, DNA, and nuclear proteins, and serves as a type of epigenetic regulation during odontoblastic differentiation (Tao et al., 2020).

Chromatin remodeling
Chromatin remodeling is a less-studied part of epigenetics but is also associated with odontogenic differentiation.ATPdependent enzymes remodel chromatin and are important for modulating chromatin structure and assembly (Ho and Crabtree, 2010).SALL1, which is expressed in pre-odontoblasts in vivo, promotes the odontoblastic differentiation of mouse dental papilla cells by activating cis-regulatory elements near Tgf-β2 and within the Runx2 locus to remodel open chromatin regions (Lin et al., 2021).Baf45a belongs to the ATPase-dependent switching defective/sucrose non-fermenting (SWI/SNF) chromatin remodeling complex.Knockdown of Baf45a leads to the downregulation of TFs that regulate odontoblast differentiation-related marker genes.It has been demonstrated that BAF45A induces remodeling of the promoters of genes that promote odontoblast differentiation in a transcriptional manner (Busby et al., 2021).(Price et al., 1998a;Price et al., 1998b).
Choi et al. developed transgenic mice expressing MT-DLX3 and observed evident dentin defects and enlarged unmineralized pulp in patients with TDO.MT-DLX3 has been demonstrated to affect odontoblastic differentiation, resulting in increased odontoblast apoptosis and distortion of dentin tubule production and dentin matrix formation, thereby downregulating dentin formation and taurodontism appeared (Choi et al., 2010).

Clinical features 4.1.2.1 Systematic features
TDO is an autosomal dominant (AD) condition characterized by anomalies in hair, teeth, and bones.TDO patients have kinky, curly hair which is featured and distinguished (Wright et al., 1997).Patients suffer from obliteration of the diploe and a lack of visible mastoid pneumatization (Price et al., 1998b).Bone density increases in the long bones, vault, base of the skull, and mastoid process, which may result from cortical sclerosis (Crawford and Aldred, 1990;Islam et al., 2005).Dolichocephaly, caused by the early closure of cranial sutures and shortened mandibles, is also a skeletal feature of TDO (Crawford and Aldred, 1990).However, phenotypic heterogeneity exists among TDO patients, which may be due to environmental or genetic factors (Jain et al., 2017).For example, curly hair at birth and dental defects such as taurodontism and enamel hypoplasia may vary from person to person clinically (Wright et al., 1997).

Dental features
Dental features including thin enamel, thin dentin, and taurodontism may be the most distinct characteristics of TDO patients (Wright et al., 1994), including thin enamel, thin dentin, and taurodontism.The teeth of patients with TDO exhibit generalized thin and/or pitted enamel hypoplasia, enlarged pulp chambers, and defective dentin.Moreover, taurodontism is commonly observed in the molars (Wright et al., 1994;Wright et al., 1997;Price et al., 1998b;Nieminen et al., 2011).Attrition and dental abscesses are also frequently observed (Crawford and Aldred, 1990).Both the primary and secondary teeth are affected, as they are smaller and spaced (Crawford and Aldred, 1990).Jain et al. also reported the precocious eruption of the permanent molars (Jain et al., 2017).

Dental management
Patients with TDO mainly suffer from dental hypersensitivity, attrition, loss of tooth structure, dental abscesses, esthetic problems, and psychosocial problems (Al-Batayneh, 2012).A comprehensive treatment plan is required to achieve a satisfactory long-term prognosis.
Restorative treatments are required for patients with TDO to recover their tooth shape.Full crowns (prefabricated stainless steel crowns) are beneficial for TDO patients (Fazel et al., 2021), as they can decrease the risk of dental caries and recover the occlusal vertical dimension.For young patients, temporary treatment, such as partial or complete overdentures, may be a potential choice since overdentures can prevent bone loss to prepare the patient for future definitive treatments (Fazel et al., 2021).Meanwhile, patients with TDO are likely to suffer from pulpal disease when the apex is open because teeth with weak enamel and dentin are susceptible to caries and attrition, resulting in pulpal exposure and an early need for endodontic treatment (Fazel et al., 2021).Patients with taurodonts are recommended vital pulp therapy instead of full pulp extirpation (Fazel et al., 2021).Furthermore, careful exploration of additional orifices and canals using magnification can increase the success rate of endodontic treatment (Jafarzadeh et al., 2008).
4.2 X-linked hypophosphatemic rickets (XLH)  (Mitchell and Mitchell, 1957).XLH (OMIM 307800), the most frequent form with a prevalence of 1:20000-60000, is a genetic disease characterized by defective mineralization of bones and tooth dentin, such as osteomalacia and radiolucent dentin (Winters et al., 1958;Opsahl Vital et al., 2012;Salmon et al., 2014;Baroncelli and Mora, 2021).It is caused by mutations in a phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) on chromosome Xp22.1-22.2(Gaucher et al., 2009).PHEX regulates FGF23 expression whereas high FGF23 concentration in serum results in hypophosphatemia and low concentration of 1,25dihydroxyvitamin D by damaging renal reabsorption of phosphate and 1a-hydroxylase activity, as well as increasing the activity of renal 24-hydroxylase (Baroncelli and Mora, 2021).Furthermore, a lack of functional PHEX causes an unnatural increase in the acidic serine-and aspartate-rich motif (ASARM) peptide, which is identified as a PHEX substrate and is derived from matrix extracellular phosphoglycoprotein (MEPE) (Salmon et al., 2013).The accumulation of the MEPE-derived ASARM peptide in XLH dentin results in impaired dentinogenesis.Salmon et al. cultured SHEDs with a phosphorylated ASARM peptide in vitro and implanted a phosphorylated ASARM peptide in vivo.It has been demonstrated that SHED differentiation and dentin formation are inhibited by the MEPE-derived ASARM peptide, as DSPP expression is decreased while MEPE expression is upregulated (Salmon et al., 2013).It has also been concluded that odontoblast differentiation and dentin mineralization may be impaired by increased MEPE accumulation in the tubules and matrix (Salmon et al., 2014).Therefore, the MEPE-ASARM system is a promising therapeutic target.

Clinical features 4.2.2.1 Systematic features
Patients with XLH suffer from systemic features, including rickets, reduced growth rate, short stature associated with rickets, osteomalacia, and gradual bowing deformities of the lower limbs (Carpenter, 2012;Emma et al., 2019;Haffner et al., 2019).Moreover, daily activities of patients with XLH can be influenced by pain and physical dysfunction (Carpenter et al., 2018).

Dental features
Patients with XLH usually have dental defects, including spontaneous periapical abscesses with fistulae that form without a history of trauma or dental caries, prominent pulp horns in the tooth enamel, and enlarged pulp chambers (Baroncelli et al., 2006).In a case reported by Okawa et al., dentin dysplasia of the extracted teeth of patients with XLH, including interglobular dentin, was observed on histopathological examination (Okawa et al., 2022).These observations suggest that dentin dysplasia is a hallmark of dental defects in patients with XLH and may be due to PHEX mutation.Enamel dysplasia has also been observed clinically (Souza et al., 2010).

Dental management
Dental treatment of patients with XLH primarily consists of preventive and endodontic management.Dentin dysplasia in the permanent teeth should be considered when formulating dental treatment plans (Okawa et al., 2022).Therefore, preventive treatment is critical for oral care management (Bradley et al., 2021).The dental pulp is likely to be infected by oral bacteria because of defective mineralization of dentin.Therefore, it is important to prevent pulpal infections (Okawa et al., 2022).Maintenance of oral hygiene, pit and fissure sealants, topical fluoride application, and enamel filling are recommended (Souza et al., 2010;Okawa et al., 2022).Patients with XLH should undergo dental examinations at least twice a year (Haffner et al., 2019).Early interventions may prevent serious dental problems (Souza et al., 2010).Endodontic treatment is essential when the pulp is infected.Root canal treatment (RCT) is suitable in most cases (Bradley et al., 2021).Antibiotics are also helpful for treating acute abscesses (Haffner et al., 2019).Moreover, systemic therapy is important for patients with XLH.Classical systemic treatments include phosphorus correction and administration of calcium (Baroncelli and Mora, 2021).

Etiology
RS (OMIM 259775) was first described in 1989 as a syndrome characterized by lethal osteosclerotic bone dysplasia (Raine et al., 1989).It is a rare autosomal recessive (AR) disorder with a prevalence of <1/1,000,000 (Simpson et al., 2007).In 2007, Simpson et al. identified the pathogenic variants in FAM20C (NM_020223.3) as the cause of RS (Simpson et al., 2007).In 2009, FAM20C variants were reported in children with mild RS phenotype who survived infancy.Therefore, RS can be divided into two types: lethal (LRS) and non-lethal (NLRS) (Simpson et al., 2009).To date, more than 40 variants of FAM20C have been identified in patients with LRS or NLRS (Palma-Lara et al., 2021).
FAM20C, a member of FAM20 (Liu et al., 2018a), is implicated in dentinogenesis and odontoblastic differentiation.Dentin defects and reduced levels of odontoblast differentiation markers have been observed in mouse models knock-out of Fam20c (Wang et al., 2012).Depletion of Fam20c in mouse dental mesenchymal cells leads to reduced expression of Runx2 and Osx/Sp7 as well as downregulated transcription of Dmp1 and Dspp, indicating that FAM20C positively regulates odontoblastic differentiation (Liu et al., 2018a).Furthermore, in mouse models with ablation of Fam20c, the expression of Dspp was reduced in odontoblasts from the root region, and the BMP signaling pathway was inhibited (Li et al., 2022b).(Raine et al., 1989;Palma-Lara et al., 2021).The characteristics of NLRS include mid-facial hypoplasia, a depressed nasal bridge, ocular proptosis, cerebral calcifications, osteosclerosis, microcephaly, and brain calcifications; however, patients with NLRS may vary in clinical features (Palma-Lara et al., 2021).

Dental management
LRS patients may die of respiratory failure, therefore a neonatal intensive care team is needed for the respiratory problems.Moreover, multidisciplinary management is essential for supporting growth and achieving better prognosis (Faundes et al., 2014).But few dental treatments have been reported, may be partly due to the fatality of RS.

Etiology
Hypophosphatasia (HPP) is a rare systemic genetic disorder resulting from mutations in ALPL gene (also known as TNSALP), which encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) and leads to diminished ALP activity (Whyte, 2016;Simon et al., 2018).Mutations in the ALPL gene can be found in the developing teeth, skeleton, lungs, kidneys, and liver, resulting in dental, skeletal, and extra-skeletal manifestations (Simon et al., 2018).Severe HPP can be explained by AR inheritance, whereas mild HPP can be explained by AD or AR inheritance (Whyte et al., 2015).
ALPL gene mutations also lead to defective odontoblastic differentiation, as the canonical Wnt signaling pathway can be impaired by ALPL deficiency.In DPSCs from patients with HPP, there is a decrease in p-GSK3β and active β-catenin, and the expression levels of odontoblastic marker genes, including DSPP and DMP1, are attenuated.In normal DPSCs, downregulation and upregulation of ALPL inhibited and promoted the levels of p-GSK3β and active β-catenin, respectively.Therefore, the odontoblastic differentiation capacity of DPSCs was impaired (Zhang et al., 2021a).(Whyte, 2016), and the clinical features are variable.Skeletal symptoms include bone and muscle pain, arthralgia, and fractures.Extra skeletal features such as seizures, calcifications in various tissues, and respiratory failure are present in HPP patients (Simon et al., 2018).

Dental features
Dental complications can be present in mild forms, such as childhood HPP, adult HPP and odontohypophosphatasia (Reibel et al., 2009).Children with HPP usually suffer from the premature loss of deciduous teeth resulting from dentin, cementum, alveolar bone dysplasia, or aplasia (Zhang et al., 2021a).Moreover, permanent dentition can also be affected, as large pulp chambers can be observed in the crown, dentin resorption, and impaired dentin mineralization (Olsson et al., 1996).Delayed dentin formation and enamel defects have also been reported (Reibel et al., 2009).

Dental management
In 2015, enzyme replacement therapy (asfotase alfa) was approved as a valid treatment for HPP patients (Whyte, 2016).Dental care is of great importance in management plans.Patients with HPP usually experience early exfoliation of many teeth; therefore, dentures can be helpful for recovering speech and mastication (Whyte, 2016).Furthermore, Zhang et al. suggested that systemic LiCl injections can be a promising therapy for patients with HPP, as LiCl can improve dentin mineralization, dentin mineral density, and the height and bone mass of alveolar bone in mouse models with ALPL depletion.Mechanistically, LiCl activates the canonical Wnt pathway, enhancing the differentiation of HPP DPSCs into odontoblasts (Zhang et al., 2021a).

Other genetic syndromes with dentin defects
Apart from the abovementioned syndromes, there are still systemic diseases with dentin defects (Su et al., 2023).Schimke immuno-osseous dysplasia (SIOD, OMIM 242900) and Elsahy-Waters syndrome (EWS, OMIM 211380) are discussed in this section, because their gene mutations may impair odontoblastic differentiation of DMSCs (Su et al., 2023).Although the pathological mechanism by which the gene mutations affect odontoblastic differentiation has not yet been revealed, how they influence osteoblasts or lead to skeletal deformations still poses a possibility for future research.
SIOD is a rare AR genetic syndrome resulting from bi-allelic mutations in SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1) (Boerkoel et al., 2002).SMARCAL1 encodes a protein from the sucrose non-fermenting 2 (SNF2) family, which serves as a DNA annealing helicase involved in chromatin remodeling (Yusufzai and Kadonaga, 2008), and SMARCAL1 is massively present in developing human teeth (Morimoto et al., 2012).In cultured SIOD fibroblasts, Wnt3a, BMP4, and TGF-β1 signaling is altered, which may shed light on the possible mechanism of SIOD dental anomalies (Morimoto et al., 2012).It is characterized by spondyloepiphyseal dysplasia, T cell immunodeficiency, renal dysfunction, facial dysmorphism, and dental anomalies (Schimke et al., 1971;Spranger et al., 1991;Boerkoel et al., 2000;Morimoto et al., 2012).Morimoto et al. reported that 66% of patients with SIOD and biallelic SMARCAL1 mutations had microdontia, hypodontia, or malformed molars (Morimoto et al., 2012).In a case report by da Fonseca, the panoramic film showed that both the primary and permanent teeth had bulbous crowns with marked cervical constriction, the pulp chambers were smaller or obliterated, and the roots were thinner, similar to the dental features of dentinogenesis imperfecta (DI) type II (da Fonseca, 2000).These characteristic dental anomalies facilitate the diagnosis of SIOD (Gendronneau et al., 2014).

Conclusion
Since the discovery of DPSCs in 2000 (Gronthos et al., 2000), the past two decades have witnessed the development of research on DMSCs (Sui et al., 2020).The odontoblastic differentiation of DMSCs is a critical step during dentin formation and is regulated by signaling pathways, TFs, and posttranscriptional and epigenetic regulation at the molecular level.In this review, recent achievements are summarized, and an atlas of the regulatory mechanisms provides a deep understanding of the odontoblastic differentiation of DMSCs.In addition, this review provides an overview of the etiology, clinical features, and dental management of genetic syndromes associated with dentin defects caused by impaired odontoblast differentiation, including TDO syndrome, XLH, RS, HPP, SIOD, and EWS.Therefore, a comprehensive mechanistic insight into the odontoblastic differentiation of DMSCs could shed light on the molecular mechanisms of known and Pan et al. 10.3389/fcell.2023.1174579

FIGURE 3
FIGURE 3Stages of odontoblastic differentiation of DMSCs, and related differentiation markers as well as signaling pathways.DMSCs have the potential to differentiate into pre-odontoblasts, polarizing odontoblasts, secretory odontoblasts, and mature odontoblasts.DMSCs are spindle-like mesenchymal stem cells.Pre-odontoblasts are cells that stop dividing and increase in size than DMSCs, and whose organelles and cytoskeletal components are uniformly distributed in the cytoplasm.Polarization of pre-odontoblasts begins when they are going to differentiate into functional odontoblasts.During this process, the odontoblasts establish a cylindrical shape and exhibit structural polarity.Once polarized, odontoblasts differ in functional terms and are named secretory odontoblasts.Pre-dentine starts to be secreted at this stage.During the process of dentin formation, the odontoblast process is elongated gradually as a direct extension of the cell body.The matrix accumulates as unmineralized layer (pre-dentin) and gradually mineralizes to form dentin. ALP and Runx2 are known as the markers of the early stage of odontoblastic differentiation, while COL1A, DSPP, DMP1, OCN, BSP, and Nestin are regarded as the markers of the late stage of odontoblastic differentiation.Moreover, different signaling pathways may play a major role in different stages of differentiation.Balic, Anguila, and Mina stated that early stages of odontoblast differentiation include the stages of pre-odontoblasts and prior to the expression of Dmp1 and Dspp.For example, BMP/TGF-β signaling regulates odontoblast differentiation in the early stage of tooth formation.Activation of Erk1/2 and p38 MAPK pathways contributed to TGF-β1-induced early differentiation of DPSCs.While in the late stage of odontoblastic differentiation, Wnt signaling is important for terminal odontoblast differentiation, and Wnt/β-catenin signaling help pre-odontoblasts differentiate into functional and fully differentiation odontoblasts.And JNK is required for the late-stage differentiation of odontoblasts induced by BMP2.Abbreviations: DMSCs: Dental mesenchymal stem cells; ALP: alkaline phosphatase; Runx2: Runt-related transcription factor 2; COL1A: collagen type I A; DMP1: Dentine matrix protein 1; DSPP: Dentin sialophosphoprotein; OCN: Osteocalcin; BSP: Bone sialoprotein; BMP: Bone morphogenetic protein; TGF-β: Transforming growth factor β; ERK: Extracellular-signal regulated kinase; MAPK: Mitogen-activated protein kinase; JNK: c-Jun amino-terminal kinase.
TDO syndrome (Online Mendelian Human Genetics (OMIM) database 190320) is caused by DLX3 mutations, and the c.571_ 574delGGGG mutation in DLX3 (MT-DLX3) is the most common etiologic mutation of TDO

TABLE 1
Factors regulating odontoblastic differentiation via signaling pathways.