Odontoblast Differentiation Research Articles

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.

Impact of Metformin on Odontoblastic Differentiation and Root Formation During Tooth Development of Diabetic Rat’s Offspring

Impact of Metformin on Odontoblastic Differentiation and Root Formation During Tooth Development of Diabetic Rat’s Offspring

Roles of SP/KLF transcription factors in odontoblast differentiation: From development to diseases.

A zinc-finger transcription factor family comprising specificity proteins (SPs) and Krüppel-like factor proteins (KLFs) plays an important role in dentin development and regeneration. However, a systematic regulatory network involving SPs/KLFs in odontoblast differentiation has not yet been described. This review examined the expression patterns of SP/KLF gene family members and their current known functions and mechanisms in odontoblast differentiation, and discussed prospective research directions for further exploration of mechanisms involving the SP/KLF gene family in dentin development. Relevant literature on SP/KLF gene family members and dentin development was acquired from PubMed and Web of Science. We discuss the expression patterns, functions, and related mechanisms of eight members of the SP/KLF gene family in dentin development and genetic disorders with dental problems. We also summarize current knowledge about their complementary or synergistic actions. Finally, we propose future research directions for investigating the mechanisms of dentin development. The SP/KLF gene family plays a vital role in tooth development. Studying the complex complementary or synergistic interactions between SPs/KLFs is helpful for understanding the process of odontoblast differentiation. Applications of single-cell and spatial multi-omics may provide a more complete investigation of the mechanism involved in dentin development.

Chitosan-Calcium Aluminate as a Cell-homing Scaffold: Its Bioactivity Testing in a Microphysiological Dental Pulp Platform.

In vitro models of the dental pulp microenvironment have been proposed for the assessment of biomaterials, to minimise animal use in operative dentistry. In this study, a scaffold/3-D dental pulp cell culture interface was created in a microchip, under simulated dental pulp pressure, to evaluate the cell-homing potential of a chitosan (CH) scaffold functionalised with calcium aluminate (the 'CHAlCa scaffold'). This microphysiological platform was cultured at a pressure of 15 cm H2O for up to 14 days; cell viability, migration and odontoblastic differentiation were then assessed. The CHAlCa scaffold exhibited intense chemotactic potential, causing cells to migrate from the 3-D culture to its surface, followed by infiltration into the macroporous structure of the scaffold. By contrast, the cells in the presence of the non-functionalised chitosan scaffold showed low cell migration and no cell infiltration. CHAlCa scaffold bioactivity was confirmed in dentin sialophosphoprotein-positive migrating cells, and odontoblastic markers were upregulated in 3-D culture. Finally, in situ mineralised matrix deposition by the cells was confirmed in an Alizarin Red-based assay, in which the CHAlCa and CH scaffolds were adapted to fit within dentin discs. More intense deposition of matrix was observed with the CHAlCa scaffold, as compared to the CH scaffold. In summary, we present an in vitro platform that provides a simple and reproducible model for selecting and developing innovative biomaterials through the assessment of their cell-homing potential. By using this platform, it was shown that the combination of calcium aluminate and chitosan has potential as an inductive biomaterial that can mediate dentin tissue regeneration during cell-homing therapies.

Development and characterization of colloidal pNIPAM-methylcellulose microgels with potential application for drug delivery in dentoalveolar tissue engineering strategies

Incorporation of growth factors, signaling molecules and drugs can be vital for the success of tissue engineering in complex structures such as the dentoalveolar region. This has led to the development of a variety of drug release systems. This study aimed to develop pNIPAM-methylcellulose microgels with different synthesis parameters based on a 23 full factorial design of experiments for this application. Microgel properties, including volume phase transition temperature (VPTT), hydrodynamic size, drug loading and release, and cytocompatibility were systematically evaluated. The results demonstrated successful copolymerization and development of the microgels, a hydrodynamic size ranging from ∼200 to ∼500 nm, and VPTT in the range of 34–39 °C. Furthermore, loading of genipin, capable of inducing odontoblastic differentiation, and its sustained release over a week was shown in all formulations. Together, this can serve as a solid basis for the development of tunable drug-delivering pNIPAM-methylcellulose microgels for specific tissue engineering applications.

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

ObjectiveTo evaluate the regulatory effect of transcription factor EB (TFEB) on the odontoblastic differentiation of dental pulp stem cells(DPSCs) in vivo and in vitro. DesignsRNA-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. ResultsTFEB 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. ConclusionsOur results showed that TFEB promoted the odontoblastic differentiation of DPSCs and identified mitophagy and metabolic reprogramming as a positive feedback loop.

Developmental Anomalies in Human Teeth: Odontoblastic Differentiation in Hamartomatous Calcifying Hyperplastic Dental Follicles Presenting with DSP, Nestin, and HES1.

Hyperplastic dental follicles (HDFs) represent odontogenic hamartomatous lesions originating from the pericoronal tissues and are often associated with impacted or embedded teeth. These lesions may occasionally feature unique calcifying bodies, known as calcifying whorled nodules (CWNs), characterized by stromal cells arranged in a whorled or spiral fashion. CWNs are typically observed in multiple calcifying hyperplastic dental follicles or regional odontodysplasia. In our study, we examined 40 cases of HDFs, including nine instances with characteristics of CWNs, referred to as calcifying hyperplastic dental follicles (CHDFs), which are infrequently accompanied by odontodysplasia. The median ages of the HDFs and CHDFs were 16 (ranging from 3 to 66) and 15 (ranging from 11 to 50) years, respectively. The lower third molars were the most frequently affected by HDSFs and CHDFs, followed by the upper canines. A histological examination was conducted on all 40 cases, with an immunohistochemical analysis performed on 21 of them. Among the cases with CWN, nine affected a single embedded tooth, with one exception. CWNs exhibited diverse calcifications featuring sparse or entirely deposited psammoma bodies, and some displayed dentinoid formation. Immunohistochemically, the stromal cells of HDFs were frequently positive for CD56 and nestin. By contrast, CWNs were negative for CD56 but positive for nestin as well as hairy and enhancer split 1 (HES1), with a few dentin sialoprotein (DSP)-positive calcified bodies. Our results revealed that hamartomatous CHDFs can impact multiple and single-embedded teeth. CWNs composed of nestin and HES1-positive ectomesenchymal cells demonstrated the potential to differentiate into odontoblasts and contribute to dentin matrix formation under the influence of HES1. This study is the first report documenting odontoblastic differentiation in HDFs. The rare occurrence of HDFs and CHDFs contributes to limited comprehension. To prevent misdiagnosis, a better understanding of these conditions is necessary.

C5L2 CRISPR KO enhances dental pulp stem cell-mediated dentinogenesis via TrkB under TNFα-induced inflammation.

Background and Objectives: Dental caries is one of the most common human pathological conditions resulting from the invasion of bacteria into the dentin. Current treatment options are limited. In many cases, endodontic therapy leads to permanent pulp tissue loss. Dentin-pulp complex regeneration involves dental pulp stem cells (DPSCs) that differentiate into odontoblast-like cells under an inflammatory context. However, limited information is available on how DPSC differentiation processes are affected under inflammatory environments. We identified the crucial role of complement C5a and its receptor C5aR in the inflammation-induced odontoblastic DPSC differentiation. Methodology: Here, we further investigated the role of a second and controversial C5a receptor, C5L2, in this process and explored the underlying mechanism. Human DPSCs were examined during 7-, 10-, and 14-day odontogenic differentiation treated with TNFα, C5L2 CRISPR, and tyrosine receptor kinase B (TrkB) antagonist [cyclotraxin-B (CTX-B)]. Results: Our data demonstrate that C5L2 CRISPR knockout (KO) enhances mineralization in TNFα-stimulated differentiating DPSCs. We further confirmed that C5L2 CRISPR KO significantly enhances dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) expression after 14-day odontoblastic DPSC differentiation, and treatment with CTX-B abolished the TNFα/C5L2 CRISPR KO-induced DSPP and DMP-1 increase, suggesting TrkB's critical role in this process. Conclusion and Key applications: Our data suggest a regulatory role of C5L2 and TrkB in the TNFα-induced odontogenic DPSC differentiation. This study may provide a useful tool to understand the mechanisms of the role of inflammation in dentinogenesis that is required for successful DPSC engineering strategies.

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

ObjectiveNotum 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. DesignThe 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/β-catenin signaling pathway were studied using alkaline phosphatase staining, alizarin red staining, quantitative polymerase chain reaction, and western blot. ResultsNOTUM was observed in the apical papilla of human tooth germs. During in vitro odontoblastic differentiation of hSCAPs, NOTUM expression initially increased, while the WNT/β-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/β-catenin pathway. However, applying the protein solely in the first 3 days enhanced odontoblastic differentiation and up-regulated the WNT/β-catenin pathway. ConclusionNOTUM demonstrated a bidirectional impact on in vitro odontoblastic differentiation of hSCAPs, potentially mediated by the WNT/β-catenin pathway. These findings suggest its promising potential for pulp-dentin complex regeneration.

Effect of composite resin monomer BisGMA on odontogenic differentiation of human dental pulp stem cells

Objective : To study the effect of composite resin monomer BisGMA on human dental pulp stem cell (hDPSCs) odontoblasts differentiation, and explore the effect of composite resin filling teeth on dentin formation. Material and Methods : separate and culture hDPSCs, identify cell phenotype by flow cytometry, and induce staining to assess multidirectional differentiation potential. The hDPSCs was stimulated with different concentrations of monomeric BisGMA, and the odontoblast differentiation capacity of each group was determined by CCK-8 assay, alkaline phosphatase activity detection, and q-PCR. Results: CCK-8 showed that the addition of 0.5 mM —— 8 mM BisGMA affected the cell increment, but no significant difference; the concentration of 0.5 mM —— 8 mM BisGMA reduced the cellular alkaline phosphatase activity and further decreased the activity with increasing concentration.The q-PCR results also showed that monomeric BisGMA inhibited the dentin-related gene OC (osteocalcin), DSPP (dentin sialophosphoprotein)Expression, and with the concentration increases, the inhibition is more obvious of. Conclusion : Composite resin monomer BisGMA has a negative effect on odontogenic differentiation of human dental pulp stem cells

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.

Chemokine‑like receptor 1‑positive cells are present in the odontoblast layer in tooth tissue in rats and humans.

Cluster of differentiation (CD)44 is a marker of dental pulp stem cells and is involved in odontoblast differentiation and calcification. Chemokine-like receptor 1 (CMKLR1), also known as chemerin receptor 23 (ChemR23) is also expressed in odontoblasts and dental pulp stem cells and is involved in inflammation suppression and tooth regeneration. Resolvin E1, a bioactive lipid, is a CMKLR1 ligand that mediates the chemerin-CMKLR1 interaction and suppresses pulpal inflammation. The present study clarified the intracellular and tissue localization of CD44 and CMKLR1 by immunohistochemical staining of normal pulp and pulp with pulpitis from 12-week-old male Wistar rat teeth or human teeth. In addition, the localization of CD44 and CMKLR1 in human dental pulp stem cells was observed by immunofluorescence staining. The present study also examined the involvement of resolvin E1 in inhibiting inflammation and calcification by western blotting. CD44- and CMKLR1-positive cells were confirmed in the odontoblast layer in normal dental pulp of rats and humans. CD44 was mainly localized in the cell membrane and CMKLR1 was mainly found in the cytoplasm of human dental pulp stem cells. CMKLR1 was also confirmed in the odontoblast layer in rats and humans with pulpitis but CD44 was not present. Following treatment of dental pulp stem cells with lipoteichoic acid, which imitates Gram-positive bacterial infection, resolvin E1 did not suppress the expression of cyclooxygenase-2 or of the odontoblast differentiation marker, dentin sialophosphoprotein. Furthermore, resolvin E1 induced the differentiation of dental pulp stem cells into odontoblasts even in the presence of the inflammatory stimulus.

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.

Physiologic dentin regeneration: its past, present, and future perspectives.

Regenerative dentistry has rapidly progressed since the advancement of stem cell biology and material science. However, more emphasis has been placed on the success of tissue formation than on how well the newly generated tissue retains the original structure and function. Once dentin is lost, tertiary dentinogenesis can be induced by new odontoblastic differentiation or re-activation of existing odontoblasts. The characteristic morphology of odontoblasts generates the tubular nature of dentin, which is a reservoir of fluid, ions, and a number of growth factors, and protects the inner pulp tissue. Therefore, understanding the dynamic but delicate process of new dentin formation by odontoblasts, or odontoblast-like cells, following dentinal defects is crucial. In this regard, various efforts have been conducted to identify novel molecules and materials that can promote the regeneration of dentin with strength and longevity. In this review, we focus on recent progress in dentin regeneration research with biological molecules identified, and discuss its potential in future clinical applications.

A leptin-loaded poly-ϵ-caprolactone 3D printing scaffold for odontoblastic differentiation in human dental pulp cells

This study investigated the effects on odontoblast differentiation of a 3D-printed poly-ϵ-caprolactone (PCL) scaffold that incorporated leptin. Material extrusion-type 3D printing with a 43 000-molecular weight PCL material was used to fabricate a PCL scaffold with a 6 mm diameter, 1 mm height, and 270–340 µm pore size. The experimental groups were PCL scaffolds (control group), PCL scaffolds with aminated surfaces (group A), and PCL scaffolds with leptin on the aminated surface (group L). The aminated surface was treated with 1,6-hexanediamine and verified by ninhydrin analysis. Leptin loading was performed using Traut’s reagent and 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC). Groups A and L showed significantly higher surface wettability, pulp cell adhesion, and proliferation than the control group. Group L exhibited increased alkaline phosphatase, calcification deposits, and mRNA and protein expression of dentin sialophosphoprotein and dentin matrix acidic phosphoprotein 1 compared with the control group. In this study, a 3D-printed PCL scaffold containing leptin was enhanced odontoblast differentiation and dental pulp cells adhesion and proliferation.

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.

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

ABSTRACT 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.

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.

Corrigendum: Single cell RNA sequencing reveals human tooth type identity and guides in vitro hiPSC derived odontoblast differentiation (iOB)

Corrigendum: Single cell RNA sequencing reveals human tooth type identity and guides in vitro hiPSC derived odontoblast differentiation (iOB)

On-demand chlorine dioxide solution enhances odontoblast differentiation through desulfation of cell surface heparan sulfate proteoglycan and subsequent activation of canonical Wnt signaling.

Heparan sulfate proteoglycans (HSPGs) surround the surface of odontoblasts, and their modification affects their affinity for Wnt ligands. This study proposes applying Matching Transformation System® (MA-T), a novel chlorinated oxidant, to enhance dentinogenesis. MA-T treatment in odontoblasts decreased sulfation of HSPG and upregulated the expression of dentin sialophosphoprotein (Dspp) and Dentin Matrix Protein 1 (Dmp1) via activation of canonical Wnt signaling in vitro. Ex vivo application of MA-T also enhanced dentin matrix formation in developing tooth explants. Reanalysis of a public single-cell RNA-seq dataset revealed significant Wnt activity in the odontoblast population, with enrichment for Wnt10a and Wnt6. Silencing assays showed that Wnt10a and Wnt6 were redundant in inducing Dspp and Dmp1 mRNA expression. These Wnt ligands' expression was upregulated by MA-T treatment, and TCF/LEF binding sites are present in their promoters. Furthermore, the Wnt inhibitors Notum and Dkk1 were enriched in odontoblasts, and their expression was also upregulated by MA-T treatment, together suggesting autonomous maintenance of Wnt signaling in odontoblasts. This study provides evidence that MA-T activates dentinogenesis by modifying HSPG and through subsequent activation of Wnt signaling.