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Fibroblast growth factor 2 (FGF2) is a crucial factor in odontoblast differentiation and dentin matrix deposition, which facilitates pulpodentin repair and regeneration. Nevertheless, the specific biological function of FGF2 in odontoblastic differentiation remains unclear because it is controlled by complex signalling pathways. This study aimed to investigate the mechanism underlying the effect of FGF2 on osteo/odontogenic differentiation of stem cells from the apical papilla (SCAP). SCAP were pretreated with conditioned media containing FGF2 for 1 week, followed by culturing in induced differentiation medium for another week. RNA sequencing (RNA-seq) combined with quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to evaluate the pathways affected by FGF2 in SCAP. Osteo/odontogenic differentiation of SCAP was determined using Alizarin red S staining, alkaline phosphatase staining, RT-qPCR, and western blotting. Pretreatment with FGF2 for 1 week increased the osteo/odontogenic differentiation ability of SCAP. RNA-seq and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that phosphatidylinositol 3-kinase (PI3K)/AKT signalling is involved in the osteogenic function of FGF2. RT-qPCR results indicated that SCAP expressed FGF receptors, and western blotting showed that p-AKT was reduced in FGF2-pretreated SCAP. The activation of the PI3K/AKT pathway partially reversed the stimulatory effect of FGF2 on osteo/odontogenic differentiation of SCAP. Our findings suggest that pretreatment with FGF2 enhances the osteo/odontogenic differentiation ability of SCAP by inhibiting the PI3K/AKT pathway., (© 2024. The Author(s).)

Odontoblast differentiation is a key process in dentin formation. Mouse dental papilla cells (mDPCs) are pivotal in dentinogenesis through their differentiation into odontoblasts. Odontoblast differentiation is intricately controlled by transcription factors (TFs) in a spatiotemporal manner. Previous research explored the role of RUNX2 and KLF4 in odontoblast lineage commitment, respectively. Building on bioinformatics analysis of our previous ATAC-seq profiling, we hypothesized that KLF4 potentially collaborates with RUNX2 to exert its biological role. To investigate the synergistic effect of multiple TFs in odontoblastic differentiation, we first examined the spatiotemporal expression patterns of RUNX2 and KLF4 in dental papilla at the bell stage using immunostaining techniques. Notably, RUNX2 and KLF4 demonstrated colocalization in preodontoblast. Further, immunoprecipitation and proximity ligation assays verified the interaction between RUNX2 and KLF4 in vitro. Specifically, the C-terminus of RUNX2 was identified as the interacting domain with KLF4. Functional implications of this interaction were investigated using small hairpin RNA-mediated knockdown of Runx2, Klf4, or both. Western blot analysis revealed a marked decrease in DSPP expression, an odontoblast differentiation marker, particularly in the double knockdown condition. Additionally, alizarin red S staining indicated significantly reduced mineralized nodule formation in this group. Collectively, our findings highlight the synergistic interaction between RUNX2 and KLF4 in promoting odontoblast differentiation from mDPCs. This study contributes to a more comprehensive understanding of the regulatory network of TFs governing odontoblast differentiation., (© 2024 Wiley Periodicals LLC.)

Background: Circular RNA (circRNA) is a key player in regulating the multidirectional differentiation of stem cells. Previous research by our group found that the blue light-emitting diode (LED) had a promoting effect on the osteogenic/odontogenic differentiation of human stem cells from apical papilla (SCAPs). This research aimed to investigate the differential expression of circRNAs during the osteogenic/odontogenic differentiation of SCAPs regulated by blue LED., Materials and Methods: SCAPs were divided into the irradiation group (4 J/cm 2 ) and the control group (0 J/cm 2 ), and cultivated in an osteogenic/odontogenic environment. The differentially expressed circRNAs during osteogenic/odontogenic differentiation of SCAPs promoted by blue LED were detected by high-throughput sequencing, and preliminarily verified by qRT-PCR. Functional prediction of these circRNAs was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the circRNA-miRNA-mRNA networks were also constructed., Results: It showed 301 circRNAs were differentially expressed. GO and KEGG analyses suggested that these circRNAs were associated with some signaling pathways related to osteogenic/odontogenic differentiation. And the circRNA-miRNA-mRNA networks were also successfully constructed., Conclusion: CircRNAs were involved in the osteogenic/odontogenic differentiation of SCAPs promoted by blue LED. In this biological process, circRNA-miRNA-mRNA networks served an important purpose, and circRNAs regulated this process through certain signaling pathways., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Methyltransferase-like 3 (METTL3) is a crucial element of N6-methyladenosine (m 6 A) modifications and has been extensively studied for its involvement in diverse biological and pathological processes. In this study, we explored how METTL3 affects the differentiation of stem cells from the apical papilla (SCAPs) into odonto/osteoblastic lineages through gain- and loss-of-function experiments. The m 6 A modification levels were assessed using m 6 A dot blot and activity quantification experiments. In addition, we employed Me-RIP microarray experiments to identify specific targets modified by METTL3. Furthermore, we elucidated the molecular mechanism underlying METTL3 function through dual-luciferase reporter gene experiments and rescue experiments. Our findings indicated that METTL3 +/- mice exhibited significant root dysplasia and increased bone loss. The m 6 A level and odonto/osteoblastic differentiation capacity were affected by the overexpression or inhibition of METTL3. This effect was attributed to the acceleration of pre-miR-665 degradation by METTL3-mediated m 6 A methylation in cooperation with the "reader" protein YTHDF2. Additionally, the targeting of distal-less homeobox 3 (DLX3) by miR-665 and the potential direct regulation of DLX3 expression by METTL3, mediated by the "reader" protein YTHDF1, were demonstrated. Overall, the METTL3/pre-miR-665/DLX3 pathway might provide a new target for SCAP-based tooth root/maxillofacial bone tissue regeneration., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

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.

Objectives: Stem cells of the apical papilla (SCAPs) provide promising candidates for dental pulp regeneration. Despite great advances in the transcriptional controls of the SCAPs fate, little is known about the regulation of SCAP differentiation., Materials and Methods: Short hairpin RNAs and full-length RNA were used to deplete or overexpress lysine demethylase 4D (KDM4D) gene expression. Western blotting, real-time RT-PCR, alizarin red staining, and scratch migration assays were used to study the role of KDM4D and the ribosomal protein encoded by RPS5 in SCAPs. RNA microarray, chromatin Immunoprecipitation (ChIP), and co-immunoprecipitation (Co-IP) assays were performed to explore the underlying molecular mechanisms., Results: KDM4D enhanced the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. The microarray results revealed that 88 mRNAs were differentially expressed in KDM4D-overexpressed SCAPs. ChIP results showed knock-down of KDM4D increased the level of H3K9me2 and H3K9me3 in CNR1 promoter region. There were 37 possible binding partners of KDM4D. KDM4D was found to combine with RPS5, which also promoted the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs., Conclusions: KDM4D promoted the osteo/dentinogenic differentiation and migration potential of SCAPs in combination with RPS5, which provides a therapeutic clue for improving SCAPs-based dental tissue regeneration., (© 2022 Wiley Periodicals LLC.)

Objective: To analyze the effect of P 11 -4 self-assembly peptide on cell viability and osteogenic capacity of SCAPs through mineral deposition and gene expression of osteogenic markers., Methods: SCAPs were seeded in contact with P 11 -4 (10 µg/ml, 100 µg/ml and 1 mg/ml) solution. Cell viability was evaluated using a colorimetric assay MTT: 3-(4,5-dimethyl-thiazolyl-2)-2,5- diphenyltetrazolium bromide) in an experimental time of 24, 48 and 72 h (n = 7). Mineral deposition and quantification provided by the cells was tested using the Alizarin Red staining and Cetylpyridinium Chloride (CPC), respectively, after 30 days (n = 4). Gene expression of Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP) and Osteocalcin (OCN) was quantified using quantitative polymerase chain reaction (RT-qPCR), at 3 and 7 days with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene, and relative gene expression was measured using the ΔΔCq method. Data were analyzed using Kruskall-Wallis followed by multiple comparisons, and T-test for gene expression with α=0.05., Results: All tested concentrations (10 µg/ml, 100 µg/ml and 1 mg/ml) were not cytotoxic at time 24 and 48 h. After 72 h, a slight decrease in cell viability was observed for the lowest concentration (10 µg/ml). The concentration of 100 µg/ml P 11 -4 showed the highest mineral deposition. However, qPCR analysis of P 11 -4 (10 µg/ml) showed upregulation of RUNX2 and OCN at 3 days, with downregulation of ALP at 3 and 7d CONCLUSION: P 11 -4 did not affect cell viability, induced mineral deposition in SCAPs, and upregulated the expression of RUNX2 and OCN genes at 3 days, while downregulating ALP expression at 3 and 7 days., Clinical Significance: Based on the results obtained in this study it can be stated that self-assembling peptide P 11 -4 is a potential candidate to induce mineralization on dental stem cells for regenerative purposes and also for a clinical use as a capping agent without compromising the cells health., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report. We certify that the submission is original work and is not under review at any other publication., (Copyright © 2023. Published by Elsevier Ltd.)

Stem cells from the apical papilla (SCAPs) are used to regulate the microenvironment of nerve defects. KDM6B, which functions as an H3K27me3 demethylase, is known to play a crucial role in neurogenesis. However, the mechanism by which KDM6B influences the neurogenesis potential of SCAPs remains unclear. We evaluated the expression of neural markers in SCAPs by using real-time RT-PCR and immunofluorescence staining. To assess the effectiveness of SCAP transplantation in the SCI model, we used the BBB scale to evaluate motor function. Additionally, toluidine blue staining and Immunofluorescence staining of NCAM, NEFM, β-III-tubulin, and Nestin were used to assess nerve tissue remodeling. Further analysis was conducted through Microarray analysis and ChIP assay to study the molecular mechanisms. Our results show that KDM6B inhibits the expression of NeuroD, TH, β-III tubulin, and Nestin. In vivo studies indicate that the SCAP-KDM6Bsh group is highly effective in restoring spinal cord structure and motor function in rats suffering from SCI. Our findings suggest that KDM6B directly binds to the HES1 promoter via regulating H3K27me3 and HES1 expression. In conclusion, our study can help understand the regulatory role of KDM6B in neurogenesis and provide more effective treatments for nerve injury.

Stem cells from the apical papilla (SCAP) are a promising resource for use in regenerative endodontic treatment (RET) that may be adversely affected by oral bacteria, which in turn can exert an effect on the success of RET. Our work aims to study the cytokine profile of SCAP upon exposure to oral bacteria and their supernatants- Fusobacterium nucleatum and Enterococcus faecalis -as well as to establish their effect on the osteogenic and immunogenic potentials of SCAP. Further, we target the presence of key proteins of the Wnt/β-Catenin, TGF-β, and NF-κB signaling pathways, which play a crucial role in adult osteogenic differentiation of mesenchymal stem cells, using the Western blot (WB) technique. The membrane-based sandwich immunoassay and transcriptomic analysis showed that, under the influence of F. nucleatum (both bacteria and supernatant), the production of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 occurred, which was also confirmed at the mRNA level. Conversely, E. faecalis reduced the secretion of the aforementioned cytokines at both mRNA and protein levels. WB analysis showed that SCAP co-cultivation with E. faecalis led to a decrease in the level of the key proteins of the Wnt/β-Catenin and NF-κB signaling pathways: β-Catenin ( p = 0.0068 *), LRP-5 ( p = 0.0059 **), and LRP-6 ( p = 0.0329 *), as well as NF-kB ( p = 0.0034 **) and TRAF6 ( p = 0.0285 *). These results suggest that oral bacteria can up- and downregulate the immune and inflammatory responses of SCAP, as well as influence the osteogenic potential of SCAP, which may negatively regulate the success of RET.

Introduction: SIRT4 is a mitochondrial sirtuin. Owing to its dependance on the cofactor nicotinamide adenine dinucleotide (NAD + ), SIRT4 can act as a mitochondrial metabolic sensor of cellular energy status. We have previously shown that enhancement of mitochondrial functions is vital for the odontogenic diff ;erentiation of dental papilla cells (DPCs) during dentinogenesis. However, whether SIRT4 serves as an effective regulator of DPC diff ;erentiation by affecting mitochondrial functions remains unexplored., Methods: Primary DPCs obtained from the first molar dental papilla of neonatal Sprague-Dawley rats were used in this study. The expression pattern of SIRT4 was observed by immunohistochemistry in the first molar of postnatal day 1 (P1) rats. The changes in SIRT4 expression during odontogenic DPC differentiation were evaluated using real-time quantitative polymerase chain reaction (PCR), western blotting, and immunofluorescence. DPCs with loss (small interfering RNA-mediated knockdown) and gain (plasmid transfection-induced overexpression) of SIRT4 function were used to explore the role of SIRT4 in odontogenic differentiation. Mitochondrial function assays were performed using ATP, reactive oxygen species (ROS), and NAD + /NADH kits to investigate the potential mechanisms involved in SIRT4-mediated dentinogenesis., Results: In the present study, we found that SIRT4 expression increased in a time-dependent manner during odontogenic differentiation bothin vivo and in vitro. Sirt4 knockdown resulted in reduced odontogenic differentiation and mineralization, whereas an opposite effect was observed with SIRT4 overexpression. Furthermore, our results verified that in addition to reducing DPC differentiation, Sirt4 knockdown could also significantly reduce ATP levels, elevate the NAD + /NADH ratio, and increase ROS levels., Conclusion: SIRT4 regulates mitochondrial functions and the antioxidant capacity of DPCs, thereby influencing dentin formation and tooth development, a phenomenon that may provide a foundation for better understanding the specific molecular mechanisms underlying dentin regeneration., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

During tooth development, dental papilla cells differentiate into odontoblasts with polarized morphology and cell function. Our previous study indicated that the C-Jun N-terminal kinase (JNK) pathway regulates human dental papilla cell adhesion, migration, and formation of focal adhesion complexes. The aim of this study was to further examine the role of the JNK pathway in dental papilla cell polarity formation. Histological staining, qPCR, and Western Blot suggested the activation of JNK signaling in polarized mouse dental papilla tissue. After performing an in vitro tooth germ organ culture and cell culture, we found that JNK inhibitor SP600125 postponed tooth germ development and reduced the polarization, migration and differentiation of mouse dental papilla cells (mDPCs). Next, we screened up-regulated polarity-related genes during dental papilla development and mDPCs or A11 differentiation. We found that Prickle3, Golga2, Golga5, and RhoA were all up-regulated, which is consistent with JNK signaling activation. Further, constitutively active RhoA mutant (RhoA Q63L) partly rescued the inhibition of SP600125 on cell differentiation and polarity formation of mDPCs. To sum up, this study suggests that JNK signaling has a positive role in the formation of dental papilla cell polarization., Competing Interests: The authors have declared that no competing interests exist.

Stem cells from apical papilla (SCAPs) are desirable sources of dentin regeneration. Epigallocatechin-3-gallate (EGCG), a natural component of green tea, shows potential in promoting the osteogenic differentiation of bone mesenchymal stem cells. However, whether EGCG regulates the odontogenic differentiation of SCAPs and how this occurs remain unknown. SCAPs from immature human third molars (16-20 years, n = 5) were treated with a medium containing different concentrations of EGCG or bone morphogenic protein 2 (BMP2), with or without LDN193189 (an inhibitor of the canonical BMP pathway). Cell proliferation and migration were analyzed using a CCK-8 assay and wound-healing assay, respectively. Osteo-/odontogenic differentiation was evaluated via alkaline phosphatase staining, alizarin red S staining, and the expression of osteo-/odontogenic markers using qPCR and Western blotting. We found that EGCG (1 or 10 μM) promoted the proliferation of SCAPs, increased alkaline phosphatase activity and mineral deposition, and upregulated the expression of osteo-/odontogenic markers including dentin sialophosphoprotein ( Dspp ), dentin matrix protein-1 ( Dmp-1 ), bone sialoprotein ( Bsp ), and Type I collagen ( Col1 ), along with the elevated expression of BMP2 and phosphorylation level of Smad1/5/9 ( p < 0.01). EGCG at concentrations below 10 μM had no significant influence on cell migration. Moreover, EGCG-induced osteo-/odontogenic differentiation was significantly attenuated via LDN193189 treatment ( p < 0.01). Furthermore, EGCG showed the ability to promote mineralization comparable with that of recombinant BMP2. Our study demonstrated that EGCG promotes the osteo-/odontogenic differentiation of SCAPs through the BMP-Smad signaling pathway.

The effects of the renin-angiotensin system (RAS) on stem cells isolated from human dental apical papilla (SCAPs) are completely unknown. Therefore, the aim of this study was to identify RAS components expressed in SCAPs and the effects of angiotensin (Ang) II and Ang-(1-7) on cell proliferation. SCAPs were collected from third molar teeth of adolescents and maintained in cell culture. Messenger RNA expression and protein levels of angiotensin-converting enzyme (ACE), ACE2, and Mas, Ang II type I (AT1) and type II (AT2) receptors were detected in SCAPs. Treatment with either Ang II or Ang-(1-7) increased the proliferation of SCAPs. These effects were inhibited by PD123319, an AT2 antagonist. While Ang II augmented mTOR phosphorylation, Ang-(1-7) induced ERK1/2 phosphorylation. In conclusion, SCAPs produce the main RAS components and both Ang II and Ang-(1-7) treatments induced cell proliferation mediated by AT2 activation through different intracellular mechanisms., (© 2020 Wiley Periodicals LLC.)

Enhancing the functions of mesenchymal stem cells (MSCs) is considered a potential approach for promoting tissue regeneration. In the present study, we investigate the role of HOXC8 in regulating differentiation and migration by using stem cells of the apical papilla (SCAPs). Our results showed that overexpression of HOXC8 suppressed the osteo-/dentinogenic differentiation, as detected by measuring alkaline phosphatase activity, in vitro mineralization, and the expressions of dentin sialophosphoprotein, dentin matrix acidic phosphoprotein 1, bone sialoprotein, runt-related transcription factor 2, and osterix in SCAPs, and inhibited in vivo osteo-/dentinogenesis of SCAPs. In addition, knockdown of HOXC8 promoted the osteo-/dentinogenic differentiation potentials of SCAPs. Mechanically, HOXC8 enhanced KDM1A transcription by directly binding to its promoter. HOXC8 and KDM1A also inhibited the migration and chemotaxis abilities of SCAPs. To sum up, HOXC8 negatively regulated the osteo-/dentinogenic differentiation and migration abilities of SCAPs by directly enhancing KDM1A transcription and indicated that HOXC8 and KDM1A could serve as potential targets for enhancing dental MSC mediated tissue regeneration., (© 2020 Wiley Periodicals, Inc.)

Background : The formation of dentin-pulp involves complex epithelial-mesenchymal interactions between Hertwig's epithelial root sheath cells (HERS) and dental papilla cells (DPCs). Earlier studies have identified some of the regulatory molecules participating in the crosstalk between HERS and DPCs and the formation of dentin-pulp. In the present study we focused on the role of HERS-secreted exosomes in DPCs and the formation of dentin-pulp. Specifically, we hypothesized that exosome-like vesicles (ELVs) might mediate the function of HERS and trigger lineage-specific differentiation of dental mesenchymal cells. To test our hypothesis, we evaluated the potential of ELVs derived from a HERS cell line (ELVs-H1) in inducing in vitro and in vivo differentiation of DPCs. Methods : ELVs-H1 were characterized using transmission electron microscopy and dynamic light scattering. The proliferation, migration, and odontoblast differentiation of DPCs after treatment with ELVs-H1, was detected by CCK8, transwell, ALP, and mineralization assays, respectively. Real time PCR and western blotting were used to detect gene and protein expression. For in vivo studies, DPC cells were mixed with collagen gel combined with or without ELVs and transplanted into the renal capsule of rats or subcutaneously into nude mice. HE staining and immunostaining were used to verify the regeneration of dentin-pulp and expression of odontoblast differentiation markers. Results : ELVs-H1 promoted the migration and proliferation of DPCs and also induced odontogenic differentiation and activation of Wnt/β-catenin signaling. ELVs-H1 also contributed to tube formation and neural differentiation in vitro . In addition, ELVs-H1 attached to the collagen gel, and were slowly released and endocytosed by DPCs, enhancing cell survival. ELVs-H1 together with DPCs triggered regeneration of dental pulp-dentin like tissue comprised of hard (reparative dentin-like tissue) and soft (blood vessels and neurons) tissue, in an in vivo tooth root slice model. Conclusion : Our data highlighted the potential of ELVs-H1 as biomimetic tools in providing a microenvironment for specific differentiation of dental mesenchymal stem cells. From a developmental perspective, these vesicles might be considered as novel mediators facilitating the epithelial-mesenchymal crosstalk. Their instructive potency might be exploited for the regeneration of dental pulp-dentin tissues., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Gestational diabetes mellitus (GDM) is an important factor involved in the pathogenesis of organ development in the offspring. Here, we analyzed the effects of GDM on odontoblastic differentiation of dental papilla cells (DPCs) and dentin formation in offspring and investigated their underlying mechanisms. A GDM rat model was induced by intraperitoneal injection of streptozotocin and offspring were collected. The results showed that GDM significantly affected odontoblast differentiation and dentin formation in offspring tooth. GDM activated the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-ĸB) signaling pathway and inhibited SMAD1/5/9 signaling to modulate the odontoblastic differentiation of DPCs in offspring. Inhibition of TLR4 signaling by treated with TAK-242 significantly reverses the suppression of odonto-differentiation of DPCs in diabetic offspring. Taken together, these data indicate GDM activated the offspring DPCs TLR4/NF-ĸB signaling, which suppressed the SMAD1/5/9 phosphorylation and then inhibited odontoblasts differentiation and dentin formation., (© 2019 Wiley Periodicals, Inc.)

Cellular differentiation is caused by highly controlled modifications in the gene expression but rarely involves a change in the DNA sequence itself. Histone acetylation is a major epigenetic factor that adds an acetyl group to histone proteins, thus altering their interaction with DNA and nuclear proteins. Illumination of the histone acetylation during dentinogenesis is important for odontoblast differentiation and dentinogenesis. In the current study, we aimed to discover the roles and regulation of acetylation at histone 3 lysine 9 (H3K9ac) and H3K27ac during dentinogenesis. We first found that both of these modifications were enhanced during odontoblast differentiation and dentinogenesis. These modifications are dynamically catalyzed by histone acetyltransferases (HATs) and deacetylases (HDACs), among which HDAC3 was decreased while p300 increased during odontoblast differentiation. Moreover, overexpression of HDAC3 or knockdown p300 inhibited odontoblast differentiation in vitro, and inhibition of HDAC3 and p300 with trichostatin A or C646 regulated odontoblast differentiation. Taken together, the results of our present study suggest that histone acetylation is involved in dentinogenesis and coordinated expression of p300- and HDAC3-regulated odontoblast differentiation through upregulating histone acetylation., (© 2019 Wiley Periodicals, Inc.)

: Stem cells isolated from the apical papilla of wisdom teeth (SCAPs) are an attractive model for tissue repair due to their availability, high proliferation rate and potential to differentiate in vitro towards mesodermal and neurogenic lineages. Adult stem cells, such as SCAPs, develop in stem cell niches in which the oxygen concentration [O 2 ] is low (3-8% compared with 21% of ambient air). In this work, we evaluate the impact of low [O 2 ] on the physiology of SCAPs isolated and processed in parallel at 21% or 3% O 2 without any hyperoxic shock in ambient air during the experiment performed at 3% O 2 . We demonstrate that SCAPs display a higher proliferation capacity at 3% O 2 than in ambient air with elevated expression levels of two cell surface antigens: the alpha-6 integrin subunit (CD49f) and the embryonic stem cell marker (SSEA4). We show that the mesodermal differentiation potential of SCAPs is conserved at early passage in both [O 2 ], but is partly lost at late passage and low [O 2 ], conditions in which SCAPs proliferate efficiently without any sign of apoptosis. Unexpectedly, we show that autophagic flux is active in SCAPs irrespective of [O 2 ] and that this process remains high in cells even after prolonged exposure to 3% O 2 ., Competing Interests: The authors declare no conflict of interest.

Biological phenotypes of mesenchymal stem cells (MSCs) are regulated by a series of biochemical elements, including microRNAs, hormones and growth factors. Our previous study illustrated a significant role of miR-141-3p during the osteogenic differentiation of stem cells from apical papilla (SCAPs). Nevertheless, the functions of miR-141-3p in regulating the proliferative ability and senescence of SCAPs have not been determined. This study identified that overexpression of miR-141-3p inhibited the proliferative ability of SCAPs. Meanwhile, the senescence of SCAPs was ahead of time. Conversely, transfection of miR-141-3p inhibitor promoted the proliferative ability of SCAPs and delayed their senescence. Yes-associated protein (YAP) was predicted as the downstream target gene of miR-141-3p by online softwares (miRDB, miRTarBase, miRWalk, and TargetScan), and was further verified by dual-luciferase reporter gene assay. Additionally, knockdown of YAP inhibited the proliferation and accelerated the senescence of SCAPs. Collectively, these findings proposed a novel direction that miR-141-3p impeded proliferative ability and promoted senescence of SCAPs through post-transcriptionally downregulating YAP., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Yunnan Baiyao is a traditional Chinese herbal remedy that has long been used for its characteristics of wound healing, bone regeneration, and anti-inflammation. However, the effects of Yunnan Baiyao on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) and the potential mechanisms remain unclear. The aim of this study was to investigate the odonto/osteogenic differentiation effects of Yunnan Baiyao on SCAPs and the underlying mechanisms involved. SCAPs were isolated and cocultured with Yunnan Baiyao conditioned media. The proliferation ability was determined by cell counting kit 8 and flow cytometry. The differentiation capacity and the involvement of NF- κ B pathway were investigated by alkaline phosphatase assay, alizarin red staining, immunofluorescence assay, real-time RT-PCR, and western blot analyses. Yunnan Baiyao conditioned medium at the concentration of 50  μ g/mL upregulated alkaline phosphatase activity, induced more mineralized nodules, and increased the expression of odonto/osteogenic genes/proteins (e.g., OCN /OCN, OPN /OPN, OSX /OSX, RUNX2 /RUNX2, ALP /ALP, COL-I /COL-I, DMP1, DSP /DSPP) of SCAPs. In addition, the expression of cytoplasmic phos-I κ B α , phos-P65, and nuclear P65 was significantly increased in Yunnan Baiyao conditioned medium treated SCAPs in a time-dependent manner. Conversely, the differentiation of Yunnan Baiyao conditioned medium treated SCAPs was obviously inhibited when these stem cells were cocultured with the specific NF- κ B inhibitor BMS345541. Yunnan Baiyao can promote the odonto/osteogenic differentiation of SCAPs via the NF- κ B signaling pathway.

Trio, the Rho guanine nucleotide exchange factor (Rho-GEF), plays diverse roles in cell migration, cell axon guidance and cytoskeleton reorganization. Conserved during evolution, Trio encodes two guanine nucleotide exchange factor domains (GEFs) and activates small GTPases. The Rho-family small GTPases RhoA and Rac1, which are target molecules of Trio, have been described to engage in craniofacial development and tooth formation. However, the exact role of Trio in tooth development remains elusive. In this study, we generated Wnt1-cre;Trio fl/fl mice to address the potential function of Trio in tooth development. Wnt1-cre;Trio fl/fl mice showed short root deformity as well as decreased expression of odontogenic makers such as RUNX2, OSX, OCN, and OPN. In vitro, Trio was silenced in human stem cells of dental papilla (SCAPs). Compared with the control group, the proliferation and migration ability in the experimental group was disrupted. After knocking down Trio in SCAPs, the cells showed phenotypes of poor odontogenic differentiation and weak mineralized nodules. To study the underlying mechanism, we investigated the p38 MAPK pathway and found that loss of Trio blocked the cascade transduction of p38 MAPK signaling. In conclusion, we identified Trio as a novel coordinator in regulating root development and clarified its relevant molecular events., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: To analyse the immunohistochemical expression of ameloblastin in the bell stage of tooth germ and compare with ameloblastoma to determine the level of differentiation of tumour cells., Study Design: This study included eleven human tooth germs with four in the early and seven in the late bell stage, and six selected archival tissue samples of ameloblastomas were studied using haematoxylin and eosin, Masson's trichrome and ameloblastin., Results: All eleven tooth germs reacted positively to ameloblastin with a characteristic inverted and sequential pattern of expression in the acellular zone of the dental papilla and enamel organ. Of the six cases of ameloblastoma, five cases showed a variable level of expression of ameloblastin in the tumour cells, whereas in one case, ameloblastin was negative in the tumour cells but positive in the stromal fibrous tissue collar., Conclusion: Expression of ameloblastin in human tooth germ is related to differentiation and mineralization, and it correlates with the state of differentiation of the tumour cells in ameloblastoma., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

MicroRNA let-7 family acts as the key regulator of the differentiation of mesenchymal stem cells (MSCs). However, the influence of let-7b on biological characteristics of stem cells from apical papilla (SCAPs) is still controversial. In this study, the expression of hsa-let-7b was obviously downregulated during the osteogenic differentiation of SCAPs. SCAPs were then infected with hsa-let-7b or hsa-let-7b inhibitor lentiviruses. The proliferation ability was determined by CCK-8 and flow cytometry. The odonto/osteogenic differentiation capacity was analyzed by alkaline phosphatase (ALP) activity, alizarin red staining, Western blot assay, and real-time RT-PCR. Bioinformatics analysis was used to screen out the target of hsa-let-7b and the target relationship was confirmed by dual luciferase reporter assay. Hsa-let-7b was of no influence on the proliferation of SCAPs. Interferential expression of hsa-let-7b increased the ALP activity as well as the formation of calcified nodules of SCAPs. Moreover, the mRNA levels of osteoblastic markers (ALP, RUNX2, OSX, OPN, and OCN) were upregulated while the protein levels of DSPP, ALP, RUNX2, OSX, OPN, and OCN also increased considerably. Conversely, overexpression of hsa-let-7b inhibited the odonto/osteogenic differentiation capacity of SCAPs. Bioinformatics analysis revealed a putative binding site of hsa-let-7b in the matrix metalloproteinase 1 (MMP1) 3'-untranslated region (3'-UTR). Dual luciferase reporter assay confirmed that hsa-let-7b targets MMP1. The odonto/osteogenic differentiation ability of SCAPs ascended after repression of hsa-let-7b, which was then reversed after co-transfection with siMMP1. Together, hsa-let-7b can suppress the odonto/osteogenic differentiation capacity of SCAPs by targeting MMP1., (© 2018 Wiley Periodicals, Inc.)

Secondary damage following spinal cord injury leads to non-reversible lesions and hampering of the reparative process. The local production of pro-inflammatory cytokines such as TNF-α can exacerbate these events. Oligodendrocyte death also occurs, followed by progressive demyelination leading to significant tissue degeneration. Dental stem cells from human apical papilla (SCAP) can be easily obtained at the removal of an adult immature tooth. This offers a minimally invasive approach to re-use this tissue as a source of stem cells, as compared to biopsying neural tissue from a patient with a spinal cord injury. We assessed the potential of SCAP to exert neuroprotective effects by investigating two possible modes of action: modulation of neuro-inflammation and oligodendrocyte progenitor cell (OPC) differentiation. SCAP were co-cultured with LPS-activated microglia, LPS-activated rat spinal cord organotypic sections (SCOS), and LPS-activated co-cultures of SCOS and spinal cord adult OPC. We showed for the first time that SCAP can induce a reduction of TNF-α expression and secretion in inflamed spinal cord tissues and can stimulate OPC differentiation via activin-A secretion. This work underlines the potential therapeutic benefits of SCAP for spinal cord injury repair.

Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta 1 (TGF-β 1 ) and TGF-β 1 -induced CTGF expression is regulated through SMAD pathway. However, there is no literature showing the expression of TGF-β 1 -SMAD2/3-CTGF signaling pathway during postnatal tooth development and the formation of junctional epithelium (JE). Hence, we aimed to analyze the localization of TGF-β 1 , CTGF and phosphorylated SMAD2/3 (p-SMAD2/3) in the developing postnatal rat molars. Wistar rats were killed at postnatal (PN) 0.5, 3.5, 7, 14 and 21days and the upper jaws were processed for immunohistochemistry. At PN0.5 and PN3.5, weak staining for TGF-β 1 and CTGF was evident in preameloblasts (PA), while moderate to strong staining was seen in odontoblasts (OD), dental papilla (DPL), secretary ameloblasts (SA), preodontoblasts (PO) and polarized odontoblasts (PoO). There was no staining for p-SMAD2/3 in PA, SA, PO and PoO, although strong staining was localized in DPL. OD was initially moderately positive and then negative for p-SMAD2/3. At PN7, intense staining for TGF-β 1 and CTGF was observed in SA, OD, dental pulp (DP) and predentin respectively. p-SMAD2/3 was strongly expressed in DP and moderately expressed in SA and OD. At PN14 and PN21, both reduced enamel epithelium (REE) and JE showed a strong reaction for TGF-β 1 and CTGF. p-SMAD2/3 was intensely and weakly expressed in REE and JE respectively. These data demonstrate that the expression of CTGF, TGF-β 1 and p-SNAD2/3 is tissue-specific and stage-specific, and indicate a regulatory role for a TGF-β 1 -SMAD2/3-CTGF signaling pathway in amelogenesis, dentinogenesis and formation of JE., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Aim: To investigate the role of Lipopolysaccharide (LPS) in the odontoclast differentiation of MDPC-23 cells. It was hypothesized that MDPC-23 odontoblast-like cells may function as odontoclasts under the influence of LPS., Methodology: MDPC-23 cells were cultured in the presence of 0.1 or 1 μg mL -1 LPS for 6 days. Cell viability was determined using the CCK8 assay. TRAP staining, dentine resorption assay and ROS detection by confocal laser scanning microscope were used to test the odontoclast-like function of the induced cells. In additional, the related protein expression was confirmed by Western blotting and ELISA. An unpaired Student's t-test and one-way anova were used in statistical analysis., Results: TRAP-positive cells, which are multinucleated, on the dentine slice were significantly increased in 1 μg mL -1 LPS-induced cells (P < 0.05). Osteoclast-specific proteins such as TRAP cathepsin K and Rac1 were upregulated in the 1 μg mL -1 LPS-treated cells (P < 0.05), whilst the expression of marker proteins of the RANKL-RANK signalling pathway (RANKL, RANK and TRAF6) in the induced cells was not significantly changed (P > 0.05). ROS production was observed in the 1 μg mL -1 LPS treatment group (P < 0.05), but no significant differences were observed in the level of RANKL in the cell supernatant between the LPS-treated group and the control group (P > 0.05)., Conclusions: A known value of 1 μg mL -1 LPS might induce odontoblast-like MDPC-23 cells to generate odontoclast-like cells or to function as odontoclasts. The data might provide a new explanation for the precursors of odontoclasts and root resorption., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Introduction: Endocyn, a pH-neutral solution of hypochlorous acid and hypochlorite has been developed for use as an endodontic irrigant. The purpose of this study was to evaluate the effect of Endocyn on human periodontal ligament (PDL) fibroblasts, rat osteosarcoma cells (UMR-106), and stem cells of the apical papilla (SCAP) compared with other commonly used endodontic irrigants., Methods: To determine cytotoxicity, cells were exposed to various concentrations of Endocyn, 6% sodium hypochlorite (NaOCl), 17% EDTA, and 2% chlorhexidine for 10 minutes, 1 hour, or 24 hours. Cell survival was measured fluorescently using calcein AM. Endocyn also was tested for its ability to inhibit SCAP proliferation and alkaline phosphatase activity. Finally, SCAP transcript expression was examined via reverse-transcriptase polymerase chain reaction., Results: Endocyn was no more toxic to PDL and UMR cells than water for up to 24 hours. Endocyn concentrations of 50% were toxic to SCAP after 1 hour of exposure. Endocyn concentrations of >20% inhibited SCAP proliferation, whereas concentrations of ≥10% inhibited alkaline phosphatase activity. Exposure of SCAP to 10% Endocyn for 3 days did not alter most transcript expression, but did significantly reduce the expression of alkaline phosphatase, fibromodulin, and osteomodulin., Conclusion: Endocyn was significantly less cytotoxic to PDL, UMR-106, and SCAP cells compared with other commonly used endodontic irrigants. High concentrations of Endocyn did inhibit some transcript expression and alkaline phosphatase activity, indicating a potential reduction in the osteogenic potential of stems cells exposed to Endocyn., (Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The disturbance of cellular attachment to dentin by sodium hypochlorite (NaOCl) may hamper pulp tissue regeneration. The aims of this study were to examine the recovering effect of EDTA on the attachment/differentiation of stemlike cells and to address the mechanisms of EDTA-induced recovery under the hypothesis that attachment to the exposed dentin matrix and the subsequent activation of integrin/phosphatidylinositol 3-kinase (PI3K) signaling play a crucial role., Methods: Mouse dental papilla (MDP) cells were cultured on bovine dentin disks treated with NaOCl (0%, 1.5%, or 6%) followed by EDTA (0%, 3%, or 17%). Cell attachment was evaluated by cell density, viability, and scanning and transmission electron microscopy. Odonto-/osteoblastic gene expression in attached MDP cells was analyzed with or without a pan-PI3K inhibitor (LY294002) using real-time polymerase chain reaction., Results: NaOCl treatment (1.5%, 10 minutes) significantly diminished attached MDP cells (P < .00001), but EDTA treatment (3% and 17%, ≥10 minutes) of NaOCl-pretreated dentin induced a significant increase in attached cells (P < .05). Ultrastructurally, MDP cells on EDTA-treated dentin showed attachment to exposed collagen fibers. MDP cells cultured on EDTA-treated disks (with or without 1.5% NaOCl pretreatment) showed significant up-regulation of alkaline phosphatase, dentin matrix protein 1, and dentin sialophosphoprotein messenger RNAs (P < .05). Alkaline phosphatase expression was down-regulated by LY294002 (P < .05)., Conclusions: Under the present experimental conditions, 10 minutes of EDTA treatment was sufficient to recover attachment/differentiation of MDP cells on 1.5% NaOCl-pretreated dentin. EDTA-induced exposure of collagen fibers and subsequent activation of integrin/PI3K signaling may contribute, at least partly, to the recovery., (Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Mesenchymal stem cells (MSCs) are a prospective cell source for tissue regeneration due to their self-renewal abilities and potential to differentiate into different cell lineages, but the molecular mechanisms of the directed differentiation and proliferation are still unknown. Recently, multiple studies have indicated the crucial role of HOX genes in MSC differentiation and proliferation. However, the role of HOXA5 in MSCs remains unknown. Here, we investigated HOXA5 function in stem cells from the apical papilla (SCAPs). After HOXA5 depletion, the results showed a significant decrease in ALP activity and a weakened mineralization ability of SCAPs. The real-time RT-PCR results showed prominently lessened expression of OPN and BSP. The CCK8 and CFSE results displayed inhibited proliferation of SCAPs, and flow cytometry assays revealed arrested cell cycle progression at the S phase. Furthermore, we found that depletion of HOXA5 upregulated p16 INK4A and p18 INK4C and downregulated the Cyclin A. Our research demonstrated that depletion of HOXA5 inhibited osteogenic differentiation and repressed cell proliferation by arresting cell cycle progression at the S phase via p16 INK4A , p18 INK4C , and Cyclin A in SCAPs, indicating that HOXA5 has a significant role in maintaining the proliferation and differentiation potential of dental-tissue-derived MSCs., (© 2017 International Federation for Cell Biology.)

Objectives: The microenvironmental niche plays the key role for maintaining the cell functions. The stem cells from apical papilla (SCAPs) are important for tooth development and regeneration. However, there is limited knowledge about the key factors in niche for maintaining the function of SCAPs. In this study, we analyse the gene expression profiles between apical papilla tissues, SCAPs and SCAPs cell sheet to identify the key genes in SCAPs niche., Materials and Methods: Microarray assays and bioinformatic analysis were performed to screen the differential genes between apical papilla tissues and SCAPs, and SCAPs and SCAPs cell sheet. Recombinant human BMP6 protein was used in SCAPs. Then CCK-8 assay, CFSE assay, alkaline phosphatase activity, alizarin red staining, quantitative calcium analysis and real-time reverse transcriptase-polymerase chain reaction were performed to investigate the cell proliferation and differentiation potentials of SCAPs., Results: Microarray analysis found that 846 genes were up-regulated and 1203 genes were down-regulated in SCAPs compared with apical papilla tissues. While 240 genes were up-regulated and 50 genes were down-regulated in SCAPs compared to in SCAPs cell sheet. Moreover, only 31 gene expressions in apical papilla tissues were recovered in cell sheet compared with SCAPs. Bioinformatic analysis identified that TGF-β, WNT and MAPK signalling pathways may play an important role in SCAPs niche. Based on the analysis, we identified one key growth factor in niche, BMP6, which could enhance the cell proliferation, the osteo/dentinogenic, neurogenic and angiogenic differentiation potentials of SCAPs., Conclusions: Our results provided insight into the mechanisms of the microenvironmental niche which regulate the function of SCAPs, and identified the key candidate genes in niche to promote mesenchymal stem cells-mediated dental tissue regeneration., (© 2017 John Wiley & Sons Ltd.)

Klf5, a member of the Krüppel-like transcription factor family, has essential roles during embryonic development, cell proliferation, differentiation, migration and apoptosis. This study was to define molecular mechanism of Klf5 during the odontoblastic differentiation. The expression of Klf5, odontoblast-differentiation markers, Dspp and Dmp1 was co-localized in odontoblastic cells at different stages of mouse tooth development and mouse dental papilla mesenchymal cells. Klf5 was able to promote odontoblastic differentiation and enhance mineral formation of mouse dental papilla mesenchymal cells. Furthermore, overexpression of Klf5 could up-regulate Dspp and Dmp1 gene expressions in mouse dental papilla mesenchymal cells. In silico analysis identified that several putative Klf5 binding sites in the promoter and first intron of Dmp1 and Dspp genes that are homologous across species lines. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis indicated that Klf5 bound to these motifs in vitro and in intact cells. The responsible regions of Dmp1 gene were located in the promoter region while effect of Klf5 on Dspp activity was in the first intron of Dspp gene. Our results identify Klf5 as an activator of Dmp1 and Dspp gene transcriptions by different mechanisms and demonstrate that Klf5 plays a pivotal role in odontoblast differentiation.

The synchronization of cell proliferation and cytodifferentiation between dental epithelial and mesenchymal cells is required for the morphogenesis of teeth with the correct functional shapes and optimum sizes. Epiprofin (Epfn), a transcription factor belonging to the Sp family, regulates dental epithelial cell proliferation and is essential for ameloblast and odontoblast differentiation. Epfn deficiency results in the lack of enamel and ironically the formation of extra teeth. We investigated the mechanism underlying the functions of Epfn in tooth development through the creation of transgenic mice expressing Epfn under the control of an epithelial cell-specific K5 promoter (K5-Epfn). We found that these K5-Epfn mice developed abnormally shaped incisors and molars and formed fewer molars in the mandible. Remarkably, ameloblasts differentiated ectopically and enamel was formed on the lingual side of the K5-Epfn incisors. By contrast, ameloblasts and enamel were found only on the labial side in wild-type mice, as Follistatin (Fst) expressed in the lingual side inhibits BMP4 signaling necessary for ameloblast differentiation. We showed that Epfn transfection into the dental epithelial cell line SF2 abrogated the inhibitory activity of Fst and promoted ameloblast differentiation of SF2 cells. We found that Epfn induced FGF9 in dental epithelial cells and this dental epithelial cell-derived FGF9 promoted dental mesenchymal cell proliferation via the FGF receptor 1c (FGFR1c). Taken together, these results suggest that Epfn preserves the balance between cell proliferation and cytodifferentiation in dental epithelial and mesenchymal cells during normal tooth development and morphogenesis. © 2016 American Society for Bone and Mineral Research., (© 2016 American Society for Bone and Mineral Research.)

Objective: Voltage-gated inward Ca 2+ currents (ICa) are triggered by cell depolarization and commonly produce transient increases in the cytoplasmic free Ca 2+ concentration. The Ca V 1.2 distal C-terminus is susceptible to proteolytic cleavage, which yields a truncated Ca V 1.2 subunit and a cleaved C-terminal fragment (CCt or DCT). Stem cells from the apical papilla (SCAPs) has a capacity for differentiation into the odontoblastic-like cells in vitro and dentin forming in vivo, which makes SCAPs advantages in tissue engineering and regenerative endodontic. The aim of this study was to investigate the effect of Ca V 1.2 and its distal C-terminal fragment in the odontoblastic differentiation of rat SCAPs (stem cells from the apical papilla)., Design: In this study, we generated stable Ca V 1.2 knockdown and DCT over-expressed rSCAPs using short hairpin RNA and DCT gene containing Lentivirus vectors, respectively. The transfected apical papilla cells were induced to differentiate into the odontoblast-like cells, and the expression of markers for odontoblastic differentiation were analyzed by alizarin red staining, Real-time Polymerase chain reaction (RT-PCR), and Western blot analysis., Results: The knockdown of Ca V 1.2 and excess expression of DCT both suppressed the expression of DSPP, ALP in mRNA level and the formation of calcium nodules., Conclusions: Our results suggest that Ca V 1.2 and DCT play important roles in the differentiation of rSCAPs, DCT might act as a transcription factor and regulate the differentiation of rSCAPs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Decellularized mammalian extracellular matrices (ECM) have been widely accepted as an ideal substrate for repair and remodelling of numerous tissues in clinical and pre-clinical studies. Recent studies have demonstrated the ability of ECM scaffolds derived from site-specific homologous tissues to direct cell differentiation. The present study investigated the suitability of hydrogels derived from different source tissues: bone, spinal cord and dentine, as suitable carriers to deliver human apical papilla derived mesenchymal stem cells (SCAP) for spinal cord regeneration. Bone, spinal cord, and dentine ECM hydrogels exhibited distinct structural, mechanical, and biological characteristics. All three hydrogels supported SCAP viability and proliferation. However, only spinal cord and bone derived hydrogels promoted the expression of neural lineage markers. The specific environment of ECM scaffolds significantly affected the differentiation of SCAP to a neural lineage, with stronger responses observed with spinal cord ECM hydrogels, suggesting that site-specific tissues are more likely to facilitate optimal stem cell behavior for constructive spinal cord regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 319-328, 2017., (© 2016 Wiley Periodicals, Inc.)

Insulin-like growth factor-1 (IGF-1) and its receptor IGF-1R play a paramount role in tooth/bone formation while hsa-let-7c actively participates in the osteogenic differentiation of mesenchymal stem cells. However, the interaction between IGF-1/IGF-1R and hsa-let-7c on the committed differentiation of stem cells from apical papilla (SCAPs) remains unclear. In this study, human SCAPs were isolated and treated with IGF-1 and hsa-let-7c over/low-expression viruses. The odonto/osteogenic differentiation of these stem cells and the involvement of mitogen-activated protein kinase (MAPK) pathway were subsequently investigated. Alizarin red staining showed that hsa-let-7c low-expression can significantly promote the mineralization of IGF-1 treated SCAPs, while hsa-let-7c over-expression can decrease the calcium deposition of IGF-1 treated SCAPs. Western blot assay and real-time reverse transcription polymerase chain reaction further demonstrated that the expression of odonto/osteogenic markers (ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, COL-I/COL-I, DSPP/DSP, and DMP-1/DMP-1) in IGF-1 treated SCAPs were significantly upregulated in Let-7c-low group. On the contrary, hsa-let-7c over-expression could downregulate the expression of these odonto/osteogenic markers. Moreover, western blot assay showed that the JNK and p38 MAPK signaling pathways were activated in Let-7c-low SCAPs but inhibited in Let-7c-over SCAPs. Together, the IGF-1/IGF-1R/hsa-let-7c axis can control the odonto/osteogenic differentiation of IGF-1-treated SCAPs via the regulation of JNK and p38 MAPK signaling pathways.

Aim: To investigate the in vitro effects of TNF-α and IL-1β on the osteogenic/dentinogenic differentiation of stem cells from the apical papilla (SCAP)., Methodology: SCAPs treated with 10 ng mL(-1) TNF-α and/or 5 ng mL(-1) IL-1β were defined as the treatment groups, with untreated cells defined as the control group. Cell proliferation was measured using the MTT assay and cell cycle analysis. Cells in all of the groups were cultured in the osteogenic/dentinogenic differentiation medium and processed for analysis at days 3, 7 and 14. The Alizarin Red S assay, alkaline phosphatase staining and real-time PCR were used to evaluate the differentiation capacity. Differences between the treatment groups and the control groups were analysed statistically using one-way anova and Dunnett's post-test., Results: Compared with the control group, the treated SCAPs were associated with a significantly greater proliferation activity at days 3 and 5 but reduced proliferation at day 9 (P < 0.05). Significantly lower calcium deposition and ALP activity were observed in the treatment groups at day 14 (P < 0.05), as well as reduced ALP, DSPP and DMP-1 expression levels (P < 0.05); however, treated SCAPs had significantly higher levels of DSPP and DMP-1 at day 7 (P < 0.05). In the control group, the ALP, DSPP and DMP-1 expression levels increased significantly from day 3 to day 14 (P < 0.05), but the treatment groups were not associated with increased expression from day 7 to day 14 (P > 0.05)., Conclusions: The pro-inflammatory cytokines TNF-α and IL-1β inhibited mineralization and osteogenic-/dentinogenic-related gene expression in SCAPs in vitro after long-term culturing. However, these cytokines induced cell proliferation and mineralization in short-term culture probably as a protective response. These findings help to elucidate the impact of inflammation on tooth development., (© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Objectives: Dental mesenchymal stem cells (MSCs) are easily obtained; however, mechanisms underlying directed differentiation of these cells remains unclear. Wnt/β-catenin signalling is essential for mesenchymal cell commitment and differentiation, and Wnt inhibition is linked to stem cell maintenance and function. Secreted frizzled-related protein 2 (SFRP2) competes with the Frizzled receptor for direct binding to Wnt and blocks activation of Wnt signalling. Here, we used stem cells derived from apical papillae (SCAPs) to study the functions of SFRP2., Materials and Methods: SCAPs were isolated from apical papillae of immature third molars. The cells were analysed using alkaline phosphatase activity assays, Alizarin red staining and quantitative calcium measurements. In addition, we evaluated expression profile of genes associated with osteogenesis and dentinogenesis (osteo-/dentinogenesis), and conducted in vivo transplantation experiments to determine osteo-/dentinogenic differentiation potential of SCAPs. ChIP assays were used to detect histone methylation at the SFRP2 promoter., Results: We found that SFRP2 enhanced osteo-/dentinogenic differentiation via Osterix, a key transcription factor in SCAPs. Furthermore, silencing SFRP2 induced SCAP cell death in osteogenic-inducing medium, indicating that SFRP2 is a key factor in maintaining SCAP survival following osteo-/dentinogenic commitment. Moreover, we found that silencing KDM2A, a histone demethylase and BCL6 co-repressor, de-repressed SFRP2 transcription by increasing histone H3K4 and H3K36 methylation at the SFRP2 promoter., Conclusions: Our results have identified a new function of SFRP2 and shed new light on the molecular mechanism underlying directed differentiation of stem cells of dental origin., (© 2016 John Wiley & Sons Ltd.)

Stem cells from apical papilla (SCAP) possess clear osteo‑/odontogenic differentiation capabilities, and are regarded as the major cellular source for root dentin development. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) serve pivotal roles in the modulation of tooth development and dentin formation. However, the synergistic effects of BMP2 and VEGF on osteo‑/odontogenic differentiation of SCAP remain unclear. The current study aimed to investigate the proliferative and osteo‑/odontogenic differentiating capabilities of BMP2 and VEGF gene-co-transfected SCAP (SCAP-BMP2-VEGF) in vitro. The basic characteristics of the isolated SCAP were identified by the induction of multipotent differentiation and by flow cytometry. Lentiviral vector‑mediated gene transfection was conducted with SCAP in order to construct blank vector‑transfected SCAP (SCAP-green fluorescent protein), BMP2 gene-transfected SCAP (SCAP-BMP2), VEGF gene‑transfected SCAP (SCAP‑VEGF) and SCAP-BMP2-VEGF. The Cell Counting Kit 8 assay was used to analyze the proliferative capacities of the four groups of cells. The expression of osteo-/odontogenic genes and proteins in the cells were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. The mineralized nodules formed by the four group cells were visualized by alkaline phosphatase (ALP) staining. Among the four groups of cells, SCAP‑VEGF was demonstrated to exhibit increased proliferation, and SCAP‑BMP2‑VEGF exhibited reduced proliferation during eight days observation. SCAP‑BMP2‑VEGF exhibited significantly increased expression levels of ALP, osteocalcin, dentin sialophosphoprotein, dentin matrix acidic phosphoprotein gene 1 and dentin sialoprotein than the other three groups at the majority of the time points. Furthermore, the SCAP‑BMP2‑VEGF group exhibited a significantly greater number of ALP‑positive mineralized nodules than the other groups following 16 days culture in vitro. In conclusion, lentiviral vector-mediated BMP2 and VEGF gene co-transfection significantly activated the osteo‑/odontogenic differentiation of human SCAP.

Dental caries, the most prevalent oral disease in dental patients, involves the phases of demineralization and destruction of tooth hard tissues like enamel, dentin, and cementum. Dentin is a major component of the root and is also the innermost layer that protects the tooth nerve, exposure of which results in pain. In this study, we used human stem cells from apical papilla (hSCAP), which are early progenitor cells, to examine the effects of recombinant human plasminogen activator inhibitor-1 (rhPAI-1) on odontogenic differentiation in vitro and in vivo. We demonstrated that rhPAI-1 promoted the proliferation and odontogenic differentiation of hSCAP and increased the expression levels of odontoblast-associated markers. We also observed that rhPAI-1 upregulated the expression of Smad4, nuclear factor I-C (NFI-C), Runx2, and osterix (OSX) during odontogenic differentiation. Notably, transplantation of rhPAI-1-treated hSCAP effectively induced odontoblastic differentiation and dentinal formation. And the differentiated odontoblast-like cells showed numerous odontoblast processes inserted in dentin tubules and arranged collagen fibers. Furthermore, odontoblast-associated markers were more highly expressed in the rhPAI-1-induced differentiated odontoblast-like cells compared with the control group. These markers were also more highly expressed in the newly formed dentin-like tissue of the rhPAI-1-treated group compared with the control group. Consistent with our in vitro results, the expression levels of Smad4, NFI-C, and OSX were also increased in the rhPAI-1-treated group compared with the control group. Taken together, these results suggest that rhPAI-1 promotes odontoblast differentiation and dentin formation of hSCAP, and Smad4/NFI-C/OSX may play critical roles in the rhPAI-1-induced odontogenic differentiation. Thus, dental stem cells from apical papilla combined with rhPAI-1 could lead to dentin regeneration in clinical implications.

Recent studies have shown that secretion of bioactive factors from mesenchymal stem cells (MSCs) plays a primary role in MSC-mediated therapy; especially for bone marrow-derived MSCs (BMSCs). MSCs from dental apical papilla (SCAPs) are involved in root development and may play a critical role in the formation of dentin and pulp. Bioactive factors secreted from SCAPs actively contribute to their environment; however, the SCAPs secretome remains unclear. To address this and gain a deeper understanding of the relevance of SCAPs secretions in a clinical setting, we used isobaric chemical tags and high-performance liquid chromatography with tandem mass spectrometry to profile the secretome of human SCAPs and then compared it to that of BMSCs. A total of 2,046 proteins were detected from the conditioned medium of SCAPs, with a false discovery rate of less than 1.0%. Included were chemokines along with angiogenic, immunomodulatory, antiapoptotic, and neuroprotective factors and extracellular matrix (ECM) proteins. The secreted levels of 151 proteins were found to differ by at least twofold when BMSCs and SCAPs were compared. Relative to BMSCs, SCAPs exhibited increased secretion of proteins that are involved in metabolic processes and transcription and lower levels of those associated with biological adhesion, developmental processes, and immune function. In addition, SCAPs secreted significantly larger amounts of chemokines and neurotrophins than BMSCs, whereas BMSCs secreted more ECM proteins and proangiogenic factors. These results may provide important clues regarding the molecular mechanisms associated with tissue regeneration and how they differ between cell sources.

Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages., Competing Interests: The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article., (© 2015 Wiley Periodicals, Inc.)

Fam20c is essential for the normal mineralization of dentin and bone. The generation of odontoblast and osteoblast cell lines carrying floxed Fam20c allele can offer valuable tools for the study of the roles of Fam20c in the mineralization of dentin and bone. The limited capability of the primary odontoblasts and osteoblasts to proliferate necessitates the development of odontoblast and osteoblast cell lines serving as substitutes for the study of differentiation and mineralization of the odontoblasts and osteoblasts. In this study, we established and characterized immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. The isolated primary mouse floxed Fam20c dental papilla mesenchymal cells and osteoblasts were immortalized by the infection of lentivirus containing Simian Virus 40 T-antigen (SV40 T-Ag). The immortalization of floxed Fam20c dental papilla mesenchymal cells and osteoblasts was verified by the long-term passages and genomic integration of SV40 T-Ag. The immortalized floxed Fam20c dental papilla mesenchymal and osteoblast cell lines not only proliferated at a high rate and retained the morphology of their primary counterparts, but also preserved the dentin and bone specific gene expression as the primary dental papilla mesenchymal cells and osteoblasts did. Consistently, the capability of the primary floxed Fam20c dental papilla mesenchymal cells and osteoblasts to mineralize was also inherited by the immortalized dental papilla mesenchymal and osteoblast cell lines. Thus, we have successfully generated the immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines., (© 2015 Wiley Periodicals, Inc.)

Aims: To analyze expression patterns of IGF-1, caspase-3 and HSP-70 in human incisor and canine tooth germs during the late bud, cap and bell stages of odontogenesis., Materials and Methods: Head areas or parts of jaw containing teeth from 10 human fetuses aged between 9th and 20th developmental weeks were immunohistochemically analyzed using IGF-1, active caspase-3 and HSP-70 markers. Semi-quantitative analysis of each marker's expression pattern was also performed., Results: During the analyzed period, IGF-1 and HSP-70 were mostly expressed in enamel organ. As development progressed, expression of IGF-1 and HSP-70 became more confined to differentiating tissues in the future cusp tip area, as well as in highly proliferating cervical loops. Few apoptotic bodies highly positive to active caspase-3 were observed in enamel organ and dental papilla from the cap stage onward. However, both enamel epithelia moderately expressed active caspase-3 throughout the investigated period., Conclusions: Expression patterns of IGF-1, active caspase-3 and HSP-70 imply importance of these factors for early human tooth development. IGF-1 and HSP-70 have versatile functions in control of proliferation, differentiation and anti-apoptotic protection of epithelial parts of human enamel organ. Active caspase-3 is partially involved in formation and apoptotic removal of primary enamel knot, although present findings might reflect its ability to perform other non-death functions such as differentiation of hard dental tissues secreting cells and guidance of ingrowth of proliferating cervical loops., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aim: To investigate the expression of CD56 in dog odontogenesis in order to elucidate the expression found in ameloblastomas., Materials and Methods: Immunohistochemical analysis of CD56 expression of developing dog teeth in the bud, cap and bell stages including the remnants of the dental lamina., Results: Weak CD56 expression was observed in the dental epithelium during the bud stage with intense staining of certain peripheral epithelial cells. Positive staining of epithelial cells was also observed in the cap stage with intense staining of the inner enamel epithelium at this stage. During the bell stage the staining was concentrated on the cervical loop areas. The dental papilla revealed positive staining throughout the cap and bell stages while the dental follicle stained intensely positive throughout all the phases examined. The dental lamina and Serres rests also stained positive for CD56., Conclusions: The expression of CD56 in dog odontogenic tissue varies according to the stage of tooth development. There is a positive correlation between the positive staining observed in ameloblastomas and their odontogenic cells of origin., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Stem cells from the human dental apical papilla (SCAP) can be obtained from almost all extracted wisdom teeth with an immature tooth root. Although different stem cell lines are used for studies, it remains elusive whether specific characteristics of the dental stem cell cultures such as proliferation rates or the cell differentiation potential are related to the cell source, e.g. the donor tissue of the dental apical papilla. To answer this question, we compared two independent SCAP cell lines from the same donor and compared them with a third cell line from another donor. We investigated the expression of stem cell markers, the efficiency of colony forming units, cell proliferation and the differentiation potential. Results showed particular differences for typical stem cell attributes such as stem cell marker expression, cell proliferation and the adipogenic differentiation. These differences were regardless of the donor of the cell lines. In conclusion, we suppose that stem cell characteristics of SCAP cell cultures are independent from the donor., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Odontogenesis is accomplished by reciprocal signaling between the epithelial and mesenchymal compartments. It is generally accepted that the inductive mesenchyme is capable of inducing the odontogenic commitment of both dental and non-dental epithelial cells. However, the duration of this signal in the developing dental mesenchyme and whether adult dental pulp tissue maintains its inductive capability remain unclear. This study investigated the contribution of growth factors to regulating the inductive potential of the dental mesenchyme. Human oral epithelial cells (OEs) were co-cultured with either human dental mesenchymal/papilla cells (FDPCs) or human dental pulp cells (ADPCs) under 2-dimensional or 3-dimensional conditions. Odontogenic-associated genes and proteins were detected by qPCR and immunofluorescence, respectively, and significant differences were observed between the two co-culture systems. The BMP7 and EREG expression levels in FDPCs were significantly higher than in ADPCs, as indicated by human growth factor PCR arrays and immunofluorescence analyses. OEs co-cultured with ADPCs supplemented with BMP7 and EREG expressed ameloblastic differentiation genes. Our study suggests that BMP7 and EREG expression in late bell-stage human dental papilla contributes to the inductive potential of dental mesenchyme. Furthermore, adult dental pulp cells supplemented with these two growth factors re-established the inductive potential of postnatal dental pulp tissue.

Background: Intercellular communication by the hedgehog cell signaling pathway is necessary for tooth development throughout the vertebrates, but it remains unclear which specific developmental signals control cell behavior at different stages of odontogenesis. To address this issue, we have manipulated hedgehog activity during zebrafish tooth development and visualized the results using confocal microscopy., Results: We first established that reporter lines for dlx2b, fli1, NF-κB, and prdm1a are markers for specific subsets of tooth germ tissues. We then blocked hedgehog signaling with cyclopamine and observed a reduction or elimination of the cranial neural crest derived dental papilla, which normally contains the cells that later give rise to dentin-producing odontoblasts. Upon further investigation, we observed that the dental papilla begins to form and then regresses in the absence of hedgehog signaling, through a mechanism unrelated to cell proliferation or apoptosis. We also found evidence of an isometric reduction in tooth size that correlates with the time of earliest hedgehog inhibition., Conclusions: We hypothesize that these results reveal a previously uncharacterized function of hedgehog signaling during tooth morphogenesis, regulating the number of cells in the dental papilla and thereby controlling tooth size., (© 2015 Wiley Periodicals, Inc.)

Introduction: Dental tissue-derived mesenchymal stem cells (MSCs) are a reliable cell source for dental tissue regeneration. However, the molecular mechanisms underlying their directed differentiation remain unclear, thus limiting their use. Trimethylation of lysine 4 of histone H3 (H3K4Me3) correlates with gene activation and osteogenic differentiation. We used stem cells from apical papilla (SCAPs) to investigate the effects of genomic changes in H3K4Me3 modification at gene promoter regions on MSC osteogenic differentiation., Methods: ChIP-on-chip assays were applied to compare the H3K4Me3 profiles at gene promoter regions of undifferentiated and differentiated SCAPs. Alkaline phosphatase activity assay, alizarin red staining, quantitative analysis of calcium, the expressions of osteogenesis-related genes, and transplantation in nude mice were used to investigate the osteogenic differentiation potentials of SCAPs., Results: In differentiated SCAPs, 119 gene promoters exhibited >2-fold increases of H3K4Me3; in contrast, the promoter regions of 21 genes exhibited >2-fold decreases of H3K4Me3. On the basis of enriched H3K4Me3 and up-regulated gene expression on the osteogenic differentiation of SCAPs, WDR63 may be a potential regulator for mediating SCAP osteogenic differentiation. Through gain-of-function and loss-of-function studies, we discovered that WDR63 enhances alkaline phosphatase activity, mineralization, and the expression of BSP, OSX, and RUNX2 in vitro. In addition, transplant experiments in nude mice confirmed that SCAP osteogenesis is triggered by activated WDR63., Conclusions: These results indicate that WDR63 is a positive enhancer for SCAP osteogenic differentiation and suggest that activation of WDR63 signaling might improve tissue regeneration mediated by MSCs of dental origin., (Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Melatonin regulates a variety of biological processes, which are the control of circadian rhythms, regulation of seasonal reproductive function and body temperature, free radical scavenging and so on. Our previous studies have shown that various cells exist in human and mouse tooth germs that express the melatonin 1a receptor (Mel1aR). However, little is known about the effects of melatonin on tooth development and growth. The present study was performed to examine the possibility that melatonin might exert its influence on tooth development. DP-805 cells, a human dental papilla cell line, were shown to express Mel1aR. Expression levels of mRNA for Mel1aR in DP-805 cells increased until 3 days after reaching confluence and decreased thereafter. Real-time reverse transcription-polymerase chain reaction showed that melatonin increased the expression of mRNAs for osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotin (DSPP). Melatonin also enhanced the mineralized matrix formation in DP-805 cell cultures in a dose-dependent manner. These results strongly suggest that melatonin may play a physiological role in tooth development/growth by regulating the cellular function of odontogenic cells in tooth germs.