Evaluation of dental pulp stem cells response to flowable nano-hybrid dental composites: A comparative analysis.

BACKGROUND: Flowable resin composites (FRC) are tooth-colored restorative materials that contain a lower filler particle content, and lower viscosity than their bulk counterparts, making them useful for specific clinical applications. Yet, their chemical makeup may impact the cellular population of the tooth pulp. This in-vitro study assessed the cytocompatibility and odontogenic differentiation capacity of dental pulp stem cells (DPSCs) in response to two recent FRC material extracts. METHODS: Extracts of the FRC Aura easyflow (AEF) and Polofil NHT Flow (PNF) were applied to DPSCs isolated from extracted human teeth. Cell viability of DPSCs was assessed using MTT assay on days 1, 3 and 7. Cell migration was assessed using the wound healing assay. DPSCs' capacity for osteo/odontogenic differentiation was assessed by measuring the degree of mineralization by Alizarin Red S staining, alkaline phosphatase enzyme (ALP) activity, and monitoring the expression of osteoprotegerin (OPG), RUNX Family Transcription Factor 2 (RUNX2), and the odontogenic marker dentin sialophosphoprotein (DSPP) by RT-PCR. Monomer release from the FRC was also assessed by High-performance liquid chromatography analysis (HPLC). RESULTS: DPSCs exposed to PNF extracts showed significantly higher cell viability, faster wound closure, and superior odontogenic differentiation. This was apparent through Alizarin Red staining of calcified nodules, elevated alkaline phosphatase activity, and increased expression of osteo/odontogenic markers. Moreover, HPLC analysis revealed a higher release of TEDGMA, UDMA, and BISGMA from AEF. CONCLUSIONS: PNF showed better cytocompatibility and enhancement of odontogenic differentiation than AEF.

Functional extracellular vesicles from SHEDs combined with gelatin methacryloyl promote the odontogenic differentiation of DPSCs for pulp regeneration.

BACKGROUND: Pulp regeneration is a novel approach for the treatment of immature permanent teeth with pulp necrosis. This technique includes the combination of stem cells, scaffolds, and growth factors. Recently, stem cell-derived extracellular vesicles (EVs) have emerged as a new methodology for pulp regeneration. Emerging evidence has proven that preconditioning is an effective scheme to modify EVs for better therapeutic potency. Meanwhile, proper scaffolding is of great significance to protect EVs from rapid clearance and destruction. This investigation aims to fabricate an injectable hydrogel loaded with EVs from pre-differentiated stem cells from human exfoliated deciduous teeth (SHEDs) and examine their effects on pulp regeneration. RESULTS: We successfully employed the odontogenic induction medium (OM) of SHEDs to generate functional EV (OM-EV). The OM-EV at a concentration of 20 µg/mL was demonstrated to promote the proliferation and migration of dental pulp stem cells (DPSCs). The results revealed that OM-EV has a better potential to promote odontogenic differentiation of DPSCs than common EVs (CM-EV) in vitro through Alizarin red phalloidin, alkaline phosphatase staining, and assessment of the expression of odontogenic-related markers. High-throughput sequencing suggests that the superior effects of OM-EV may be attributed to activation of the AMPK/mTOR pathway. Simultaneously, we prepared a photocrosslinkable gelatin methacryloyl (GelMA) to construct an OM-EV-encapsulated hydrogel. The hydrogel exhibited sustained release of OM-EV and good biocompatibility for DPSCs. The released OM-EV from the hydrogel could be internalized by DPSCs, thereby enhancing their survival and migration. In tooth root slices that were subcutaneously transplanted in nude mice, the OM-EV-encapsulated hydrogel was found to facilitate dentinogenesis. After 8 weeks, there was more formation of mineralized tissue, as well as higher levels of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1). CONCLUSIONS: The effects of EV can be substantially enhanced by preconditioning of SHEDs. The functional EVs from SHEDs combined with GelMA are capable of effectively promoting dentinogenesis through upregulating the odontogenic differentiation of DPSCs, which provides a promising therapeutic approach for pulp regeneration.

A xenogeneic extracellular matrix-based 3D printing scaffold modified by ceria nanoparticles for craniomaxillofacial hard tissue regeneration via osteo-immunomodulation.

Hard tissue engineering scaffolds especially 3D printed scaffolds were considered an excellent strategy for craniomaxillofacial hard tissue regeneration, involving crania and facial bones and teeth. Porcine treated dentin matrix (pTDM) as xenogeneic extracellular matrix has the potential to promote the stem cell differentiation and mineralization as it contains plenty of bioactive factors similar with human-derived dentin tissue. However, its application might be impeded by the foreign body response induced by the damage-associated molecular patterns of pTDM, which would cause strong inflammation and hinder the regeneration. Ceria nanoparticles (CNPs) show a great promise at protecting tissue from oxidative stress and influence the macrophages polarization. Using 3D-bioprinting technology, we fabricated a xenogeneic hard tissue scaffold based on pTDM xenogeneic TDM-polycaprolactone (xTDM/PCL) and we modified the scaffolds by CNPs (xTDM/PCL/CNPs). Through series ofin vitroverification, we found xTDM/PCL/CNPs scaffolds held promise at up-regulating the expression of osteogenesis and odontogenesis related genes including collagen type 1, Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein-2, osteoprotegerin, alkaline phosphatase (ALP) and DMP1 and inducing macrophages to polarize to M2 phenotype. Regeneration of bone tissues was further evaluated in rats by conducting the models of mandibular and skull bone defects. Thein vivoevaluation showed that xTDM/PCL/CNPs scaffolds could promote the bone tissue regeneration by up-regulating the expression of osteogenic genes involving ALP, RUNX2 and bone sialoprotein 2 and macrophage polarization into M2. Regeneration of teeth evaluated on beagles demonstrated that xTDM/PCL/CNPs scaffolds expedited the calcification inside the scaffolds and helped form periodontal ligament-like tissues surrounding the scaffolds.

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.

S100a6 knockdown promotes the differentiation of dental epithelial cells toward the epidermal lineage instead of the odontogenic lineage.

Tooth development is a complex process involving various signaling pathways and genes. Recent findings suggest that ion channels and transporters, including the S100 family of calcium-binding proteins, may be involved in tooth formation. However, our knowledge in this regard is limited. Therefore, this study aimed to investigate the expression of S100 family members and their functions during tooth formation. Tooth germs were extracted from the embryonic and post-natal mice and the expression of S100a6 was examined. Additionally, the effects of S100a6 knockdown and calcium treatment on S100a6 expression and the proliferation of SF2 cells were examined. Microarrays and single-cell RNA-sequencing indicated that S100a6 was highly expressed in ameloblasts. Immunostaining of mouse tooth germs showed that S100a6 was expressed in ameloblasts but not in the undifferentiated dental epithelium. Additionally, S100a6 was localized to the calcification-forming side in enamel-forming ameloblasts. Moreover, siRNA-mediated S100a6 knockdown in ameloblasts reduced intracellular calcium concentration and the expression of ameloblast marker genes, indicating that S100a6 is associated with ameloblast differentiation. Furthermore, S100a6 knockdown inhibited the ERK/PI3K signaling pathway, suppressed ameloblast proliferation, and promoted the differentiation of the dental epithelium toward epidermal lineage. Conclusively, S100a6 knockdown in the dental epithelium suppresses cell proliferation via calcium and intracellular signaling and promotes differentiation of the dental epithelium toward the epidermal lineage.

The role of autophagy in odontogenesis, dental implant surgery, periapical and periodontal diseases.

Autophagy is a cellular process that is evolutionarily conserved, involving the sequestration of damaged organelles and proteins into autophagic vesicles, which subsequently fuse with lysosomes for degradation. Autophagy controls the development of many diseases by influencing apoptosis, inflammation, the immune response and different cellular processes. Autophagy plays a significant role in the aetiology of disorders associated with dentistry. Autophagy controls odontogenesis. Furthermore, it is implicated in the pathophysiology of pulpitis and periapical disorders. It enhances the survival, penetration and colonization of periodontal pathogenic bacteria into the host periodontal tissues and facilitates their escape from host defences. Autophagy plays a crucial role in mitigating exaggerated inflammatory reactions within the host's system during instances of infection and inflammation. Autophagy also plays a role in the relationship between periodontal disease and systemic diseases. Autophagy promotes wound healing and may enhance implant osseointegration. This study reviews autophagy's dento-alveolar effects, focusing on its role in odontogenesis, periapical diseases, periodontal diseases and dental implant surgery, providing valuable insights for dentists on tooth development and dental applications. A thorough examination of autophagy has the potential to discover novel and efficacious treatment targets within the field of dentistry.

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

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

[Theory and practice of mesenchymal condensation directing tooth development and regeneration].

Mesenchymal stem cells, under spatiotemporal regulation of genes and microenvironment, are capable of spontaneously aggregating into dense regions, a phenomenon known as mesenchymal condensation. Mesenchymal condensation is an evolutionarily conserved developmental event that is critical in initiating morphogenesis of teeth and systemic organs. Mesenchymal stem cells hold the intrinsic ability to self-assemble in culture, and the generation of stem cell aggregates based on this property that mimics developmental mesenchymal condensation has become a potent and promising approach in regenerative medicine. This review discusses the mesenchymal condensation principles and its role as well as mechanism in tooth morphogenesis, as well as the engineering strategies for constructing mesenchymal stem cell aggregates and their application experience in tooth regeneration. It aims to start from the perspective of "development-inspired regeneration" and provide insights into understanding stem cell developmental biology and establishing new organ regenerative strategies.

[Research progress in methylation modification in tooth root development].

Methylation modification is one of the most common epigenetic modification regulation in eukaryotes, including histone methylation, DNA methylation, RNA methylation, etc., which plays an important regulatory role in physiological processes and pathologic occurrence and development. Tooth root development is carried out by both epithelial and mesenchymal cells and involves a variety of cell-molecular interactions. In recent years, a large number of studies have found that methylation plays a key role in the regulation of tooth root development and expands the mechanism network of tooth root development. In this paper, we review the role and mechanism of methylation modification during root development.

Construction of dental pulp decellularized matrix by cyclic lavation combined with mechanical stirring and its proteomic analysis.

The decellularized matrix has a great potential for tissue remodeling and regeneration; however, decellularization could induce host immune rejection due to incomplete cell removal or detergent residues, thereby posing significant challenges for its clinical application. Therefore, the selection of an appropriate detergent concentration, further optimization of tissue decellularization technique, increased of biosafety in decellularized tissues, and reduction of tissue damage during the decellularization procedures are pivotal issues that need to be investigated. In this study, we tested several conditions and determined that 0.1% Sodium dodecyl sulfate and three decellularization cycles were the optimal conditions for decellularization of pulp tissue. Decellularization efficiency was calculated and the preparation protocol for dental pulp decellularization matrix (DPDM) was further optimized. To characterize the optimized DPDM, the microstructure, odontogenesis-related protein and fiber content were evaluated. Our results showed that the properties of optimized DPDM were superior to those of the non-optimized matrix. We also performed the 4D-Label-free quantitative proteomic analysis of DPDM and demonstrated the preservation of proteins from the natural pulp. This study provides a optimized protocol for the potential application of DPDM in pulp regeneration.

Application of Slice Culture System for Successional Dental Lamina in Diphyodont Mammals.

Replacement teeth develop from the successional dental lamina (SDL). Understanding how SDL transitions from quiescence to initiation is crucial for preserving dental lamina stem cells in the jawbone microenvironment and for complete tooth regeneration. Miniature pigs are good models for studying human tooth replacement because of their similarities to humans. However, the molecular mechanisms and cellular composition that initiate SDL development remain unclear. One possible reason for this is the limitations of the current methods for culturing SDL in vitro, such as the inability to directly observe tooth morphological changes during culture and low tissue viability. This study aimed to improve the in vitro culture method for SDL. Using a McIlwain Tissue Chopper, we obtained mandibular slices containing deciduous canine and SDL of permanent canine. The slices were approximately 500 µm thick and were cultured on a Transwell membrane supported with metal grids over medium. The SDL developed into the bud stage on the second day and entered the cap stage on the fifth day in vitro. The expression of proliferation markers, cell death markers, and key odontogenetic genes in vitro was similar to that observed in vivo. In conclusion, we successfully applied a slice culture system to the SDL of miniature pigs. This slice culture method allowed us to directly visualize SDL initiation and further elucidate the molecular mechanisms underlying the initiation of permanent tooth development.

A Wnt10a-Notch signaling axis controls Hertwig's epithelial root sheath cell behaviors during root furcation patterning.

Human with bi-allelic WNT10A mutations and epithelial Wnt10a knockout mice present enlarged pulp chamber and apical displacement of the root furcation of multi-rooted teeth, known as taurodontism; thus, indicating the critical role of Wnt10a in tooth root morphogenesis. However, the endogenous mechanism by which epithelial Wnt10a regulates Hertwig's epithelial root sheath (HERS) cellular behaviors and contributes to root furcation patterning remains unclear. In this study, we found that HERS in the presumptive root furcating region failed to elongate at an appropriate horizontal level in K14-Cre;Wnt10afl/fl mice from post-natal day 0.5 (PN0.5) to PN4.5. EdU assays and immunofluorescent staining of cyclin D1 revealed significantly decreased proliferation activity of inner enamel epithelial (IEE) cells of HERS in K14-Cre;Wnt10afl/fl mice at PN2.5 and PN3.5. Immunofluorescent staining of E-Cadherin and acetyl-α-Tubulin demonstrated that the IEE cells of HERS tended to divide perpendicularly to the horizontal plane, which impaired the horizontal extension of HERS in the presumptive root furcating region of K14-Cre;Wnt10afl/fl mice. RNA-seq and immunofluorescence showed that the expressions of Jag1 and Notch2 were downregulated in IEE cells of HERS in K14-Cre;Wnt10afl/fl mice. Furthermore, after activation of Notch signaling in K14-Cre;Wnt10afl/fl molars by Notch2 adenovirus and kidney capsule grafts, the root furcation defect was partially rescued. Taken together, our study demonstrates that an epithelial Wnt10a-Notch signaling axis is crucial for modulating HERS cell proper proliferation and horizontal-oriented division during tooth root furcation morphogenesis.

Blocking endogenous retinoic acid degradation induces oral tooth formation in zebrafish.

According to Dollo's Law of irreversibility in evolution, a lost structure is usually considered to be unable to reappear in evolution due to the accumulation over time of mutations in the genes required for its formation. Cypriniform fish are a classic model of evolutionary loss because, while they form fully operational teeth in the ventral posterior pharynx, unlike other teleosts, they do not possess oral teeth. Paleontological data show that Cypriniforms, a clade of teleost fish that includes the zebrafish, lost their oral teeth 50 to 100 Mya. In order to attempt to reverse oral tooth loss in zebrafish, we block the degradation of endogenous levels of retinoic acid (RA) using a specific inhibitor of the Cyp26 RA degrading enzymes. We demonstrate the inhibition of endogenous RA degradation is sufficient to restore oral tooth induction as marked by the re-appearance of expression of early dental mesenchyme and epithelium genes such as dlx2b and sp7 in the oral cavity. Furthermore, we show that these exogenously induced oral tooth germs are able to be at least partly calcified. Taken together, our data show that modifications of signaling pathways can have a significant effect on the reemergence of once-lost structures leading to experimentally induced reversibility of evolutionary tooth loss in cypriniforms.

Deep learning methods for fully automated dental age estimation on orthopantomograms.

OBJECTIVES: This study aimed to use all permanent teeth as the target and establish an automated dental age estimation method across all developmental stages of permanent teeth, accomplishing all the essential steps of tooth determination, tooth development staging, and dental age assessment. METHODS: A three-step framework for automatically estimating dental age was developed for children aged 3 to 15. First, a YOLOv3 network was employed to complete the tasks of tooth localization and numbering on a digital orthopantomogram. Second, a novel network named SOS-Net was established for accurate tooth development staging based on a modified Demirjian method. Finally, the dental age assessment procedure was carried out through a single-group meta-analysis utilizing the statistical data derived from our reference dataset. RESULTS: The performance tests showed that the one-stage YOLOv3 detection network attained an overall mean average precision 50 of 97.50 for tooth determination. The proposed SOS-Net method achieved an average tooth development staging accuracy of 82.97% for a full dentition. The dental age assessment validation test yielded an MAE of 0.72 years with a full dentition (excluding the third molars) as its input. CONCLUSIONS: The proposed automated framework enhances the dental age estimation process in a fast and standard manner, enabling the reference of any accessible population. CLINICAL RELEVANCE: The tooth development staging network can facilitate the precise identification of permanent teeth with abnormal growth, improving the effectiveness and comprehensiveness of dental diagnoses using pediatric orthopantomograms.

Effect of Low-Level Diode Laser and Red Light-Emitting Diode on Survival and Osteogenic/Odontogenic Differentiation of Human Dental Pulp Stem Cells.

Background: This investigation set out to compare the impacts of low-level diode laser (LLDL) and red light-emitting diode (LED) on the survival of human dental pulp stem cells (hDPSCs) and osteogenic/odontogenic differentiation. Methods and materials: In this ex vivo experimental study, the experimental groups underwent the irradiation of LLDL (4 J/cm2 energy density) and red LED in the osteogenic medium. Survival of hDPSCs was assessed after 24 and 48 h (n = 9) using the methyl thiazolyl tetrazolium (MTT) assay. The assessment of osteogenic/odontogenic differentiation was conducted using alizarin red staining (ARS; three repetitions). The investigation of osteogenic and odontogenic gene expression was performed at two time points, specifically 24 and 48 h (n = 12). This analysis was performed utilizing real-time reverse-transcription polymerase chain reaction (RT-PCR). The groups were compared at each time point using SPSS version 24. To analyze the data, the Mann-Whitney U test, analysis of variance, Tukey's test, and t-test were utilized. Results: The MTT assay showed that LLDL significantly decreased the survival of hDPSCs after 48 h, compared with other groups (p < 0.05). The qualitative results of ARS revealed that LLDL and red LED increased the osteogenic differentiation of hDPSCs. LLDL and red LED both upregulated the expression of osteogenic/odontogenic genes, including bone sialoprotein (BSP), alkaline phosphatase (ALP), dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP), in hDPSCs. The LLDL group exhibited a higher level of gene upregulation (p < 0.0001). Conclusions: The cell survival of hDPSCs was reduced, despite an increase in osteogenic/odontogenic activity. Clinical relevance: Introduction of noninvasive methods in regenerative endodontic treatments.

The significant role of glycosaminoglycans in tooth development.

This review delves into the roles of glycosaminoglycans (GAGs), integral components of proteoglycans, in tooth development. Proteoglycans consist of a core protein linked to GAG chains, comprised of repeating disaccharide units. GAGs are classified into several types, such as hyaluronic acid, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate. Functioning as critical macromolecular components within the dental basement membrane, these GAGs facilitate cell adhesion and aggregation, and play key roles in regulating cell proliferation and differentiation, thereby significantly influencing tooth morphogenesis. Notably, our recent research has identified the hyaluronan-degrading enzyme Transmembrane protein 2 (Tmem2) and we have conducted functional analyses using mouse models. These studies have unveiled the essential role of Tmem2-mediated hyaluronan degradation and its involvement in hyaluronan-mediated cell adhesion during tooth formation. This review provides a comprehensive summary of the current understanding of GAG functions in tooth development, integrating insights from recent research, and discusses future directions in this field.

The ectopic mandibular canines can start tooth formation in three different locations: a case series study based on single orthopantomograms from 47 individuals.

INTRODUCTION: A former study on orthopantomograms from young children with abnormal dental development (not canine ectopia) demonstrated that the tooth bud of the mandibular canine, compared to a stable longitudinal canine axis, could be located normally, anteriorly or posteriorly, with close relation to the first premolar. AIM: The aim of the present study is to analyse on orthopantomograms if the canine axis can demonstrate where the ectopic mandibular canine started tooth formation. MATERIALS: The material consists of orthopantomograms with ectopic mandibular canines and presence of primary mandibular canines from 47 cases (29 cases 9-21 years old and 18 cases with unknown ages). The primary canines demonstrated from minor apical resorption to more severe apical resorption. METHODS: Based on canine maturity, location of the canine axes and the interrelationships between the roots of the permanent canine and first premolar, the location from where the canine started tooth formation was determined. Canine maturity. Maturity stage below half root length and maturity stage above half root length revealed that 11 ectopic canines had less than half root length and 36 cases more than half root length. Canine axes. The canine axis, through the length of the primary canines Ax, is inserted on drawings of the orthopantomograms using the tracing programme Inkscape®. Interrelationship between roots. By visual inspection, the distance between the canine and first premolar was designated close distance, normal distance and extended distance. RESULTS: The results are divided into 3 groups. Group 1: The initial site of the permanent ectopic canine is located within the canine axis (6 cases). Group 2: The initial site of the permanent ectopic canine is located posterior to the canine axis (36 cases). Group 3: The initial site of the permanent ectopic canine is located anterior to the canine axis (5 cases). CONCLUSION: The study explained that the canine axis could divide cases of ectopic canines into three groups according to the location from where tooth formation starts. For getting closer to the pattern of the ectopic canine eruption, it is necessary to analyse series of orthopantomograms taken from the same individual over several years.

Effects of Novel Nanoparticulate Bioceramic Endodontic Material on Human Dental Pulp Stem Cells In Vitro.

OBJECTIVES: This study aimed to investigate the in vitro effects of root canal filling and repair paste (nRoot BP) on human dental pulp stem cells (hDPSCs). METHODS: The effects of nRoot BP and iRoot BP Plus on the adhesion, proliferation, migration, and differentiation of hDPSCs were examined in vitro for 72 hours. The adhesion of cells was observed using immunofluorescence rhodamine ghost pen cyclic peptide staining and scanning electron microscopy (SEM). Cell density and changes in migration area were measured under a fluorescence inverted microscope. Fluorescent quantitative PCR was performed to detect genes related to odontogenesis and osteogenesis. RESULTS: Cells adhering to the surfaces of nRoot BP and iRoot BP Plus exhibited similar irregular polygonal morphologies, with cells extending irregular pseudopods to adhere to the materials. CCK-8 results indicated that the density of living cells for nRoot BP and iRoot BP Plus was lower than that of the blank control group at 3 and 5 days of culture. There was no significant difference in cell migration between the groups (P > .05). The migration ability of iRoot BP Plus and nRoot BP was similar to that of the control group. Both nRoot BP and iRoot BP Plus increased the expression of the RUNX2 gene, but there was no significant difference between the groups (P < .05). Furthermore, both nRoot BP and iRoot BP Plus downregulated the expression of the DSPP gene, with no significant difference between them (P > .05). CONCLUSIONS: nRoot BP exhibited a slight inhibition of hDPSC proliferation but did not affect the adhesion and migration of hDPSCs. The impact of nRoot BP on the osteogenic and odontogenic differentiation of hDPSCs was similar to that of iRoot BP Plus.

Regulatory role of N-myc downregulated genes in amelogenesis in rats.

Cytodifferentiation of odontogenic cells, a late stage event in odontogenesis is based on gene regulation. However, studies on the identification of the involved genes are scarce. The present study aimed to search for molecules for the cytodifferentiation of ameloblastic cells in rats. Differential display-PCR revealed a differentially expressed gene between cap/early bell stage and hard tissue formation stage in molars. This gene was identified as N-myc Downregulated Gene 1 (Ndrg1), which is the first report in tooth development. Real time PCR and western blotting confirmed that the mRNA level of Ndrg1 was higher during enamel formation than the cap stage. Ndrg1 expression was upregulated in the early bell, crown, and root stages in a time-dependent manner. These patterns of expression were similar in Ndrg2, but Ndrg3 and Ndrg4 levels did not change during the developmental stages. Immunofluorescence revealed that strong immunoreactivity against Ndrg1 were detected in differentiated ameloblasts only, not inner enamel epithelium, odontoblasts and ameloblastic cells in defected enamel regions. Alkaline phosphatase and alizarin red s stains along with real time PCR, revealed that Ndrg1 and Ndrg2 were involved in cytodifferentiation and enamel matrix mineralization by selectively regulating amelogenin and ameloblastin genes in SF2 ameloblastic cells. These results suggest that Ndrg may play a crucial functional role in the cytodifferentiation of ameloblasts for amelogenesis.

The Essential Role of Proteoglycans and Glycosaminoglycans in Odontogenesis.

Tooth development and regeneration are regulated through a complex signaling network. Previous studies have focused on the exploration of intracellular signaling regulatory networks, but the regulatory roles of extracellular networks have only been revealed recently. Proteoglycans, which are essential components of the extracellular matrix (ECM) and pivotal signaling molecules, are extensively involved in the process of odontogenesis. Proteoglycans are composed of core proteins and covalently attached glycosaminoglycan chains (GAGs). The core proteins exhibit spatiotemporal expression patterns during odontogenesis and are pivotal for dental tissue formation and periodontium development. Knockout of core protein genes Biglycan, Decorin, Perlecan, and Fibromodulin has been shown to result in structural defects in enamel and dentin mineralization. They are also closely involved in the development and homeostasis of periodontium by regulating signaling transduction. As the functional component of proteoglycans, GAGs are negatively charged unbranched polysaccharides that consist of repeating disaccharides with various sulfation groups; they provide binding sites for cytokines and growth factors in regulating various cellular processes. In mice, GAG deficiency in dental epithelium leads to the reinitiation of tooth germ development and the formation of supernumerary incisors. Furthermore, GAGs are critical for the differentiation of dental stem cells. Inhibition of GAGs assembly hinders the differentiation of ameloblasts and odontoblasts. In summary, core proteins and GAGs are expressed distinctly and exert different functions at various stages of odontogenesis. Given their unique contributions in odontogenesis, this review summarizes the roles of proteoglycans and GAGs throughout the process of odontogenesis to provide a comprehensive understanding of tooth development.