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Stem Cell Research & Therapy Jul 2023Dental pulp stem cells (DPSCs) play a crucial role in dentin-pulp complex regeneration. Further understanding of the mechanism by which DPSCs remain in a quiescent state...
BACKGROUND
Dental pulp stem cells (DPSCs) play a crucial role in dentin-pulp complex regeneration. Further understanding of the mechanism by which DPSCs remain in a quiescent state could contribute to improvements in the dentin-pulp complex and dentinogenesis.
METHODS
TSC1 conditional knockout (DMP1-Cre+; TSC1, hereafter CKO) mice were generated to increase the activity of mechanistic target of rapamycin complex 1 (mTORC1). H&E staining, immunofluorescence and micro-CT analysis were performed with these CKO mice and littermate controls. In vitro, exosomes were collected from the supernatants of MDPC23 cells with different levels of mTORC1 activity and then characterized by transmission electron microscopy and nanoparticle tracking analysis. DPSCs were cocultured with MDPC23 cells and MDPC23 cell-derived exosomes. Alizarin Red S staining, ALP staining, qRT‒PCR, western blotting analysis and micro-RNA sequencing were performed.
RESULTS
Our study showed that mTORC1 activation in odontoblasts resulted in thicker dentin and higher dentin volume/tooth volume of molars, and it increased the expression levels of the exosome markers CD63 and Alix. In vitro, when DPSCs were cocultured with MDPC23 cells, odontoblastic differentiation was inhibited. However, the inhibition of odontoblastic differentiation was reversed when DPSCs were cocultured with MDPC23 cells with mTORC1 overactivation. To further study the effects of mTORC1 on exosome release from odontoblasts, MDPC23 cells were treated with rapamycin or shRNA-TSC1 to inactivate or activate mTORC1, respectively. The results revealed that exosome release from odontoblasts was negatively correlated with mTORC1 activity. Moreover, exosomes derived from MDPC23 cells with active or inactive mTORC1 inhibited the odontoblastic differentiation of DPSCs at the same concentration. miRNA sequencing analysis of exosomes that were derived from shTSC1-transfected MDPC23 cells, rapamycin-treated MDPC23 cells or nontreated MDPC23 cells revealed that the majority of the miRNAs were similar among these groups. In addition, exosomes derived from odontoblasts inhibited the odontoblastic differentiation of DPSCs, and the inhibitory effect was positively correlated with exosome concentration.
CONCLUSION
mTORC1 regulates exosome release from odontoblasts to inhibit the odontoblastic differentiation of DPSCs, but it does not alter exosomal contents. These findings might provide a new understanding of dental pulp complex regeneration.
Topics: Mice; Animals; Odontoblasts; Extracellular Matrix Proteins; Dental Pulp; Exosomes; Cell Differentiation; Stem Cells; Cells, Cultured
PubMed: 37422687
DOI: 10.1186/s13287-023-03401-9 -
Frontiers in Physiology 2022According to the "hydrodynamic theory," dentinal pain or sensitivity is caused by dentinal fluid movement following the application of various stimuli to the dentin...
According to the "hydrodynamic theory," dentinal pain or sensitivity is caused by dentinal fluid movement following the application of various stimuli to the dentin surface. Recent convergent evidence has shown that plasma membrane deformation, mimicking dentinal fluid movement, activates mechanosensitive transient receptor potential (TRP)/Piezo channels in odontoblasts, with the Ca signal eliciting the release of ATP from pannexin-1 (PANX-1). The released ATP activates the P2X receptor, which generates and propagates action potentials in the intradental Aδ afferent neurons. Thus, odontoblasts act as sensory receptor cells, and odontoblast-neuron signal communication established by the TRP/Piezo channel-PANX-1-P2X receptor complex may describe the mechanism of the sensory transduction sequence for dentinal sensitivity. To determine whether odontoblast-neuron communication and odontoblasts acting as sensory receptors are essential for generating dentinal pain, we evaluated nociceptive scores by analyzing behaviors evoked by dentinal sensitivity in conscious Wistar rats and Cre-mediated transgenic mouse models. In the dentin-exposed group, treatment with a bonding agent on the dentin surface, as well as systemic administration of A-317491 (P2X receptor antagonist), mefloquine and PANX (non-selective and selective PANX-1 antagonists), GsMTx-4 (selective Piezo1 channel antagonist), and HC-030031 (selective TRPA1 channel antagonist), but not HC-070 (selective TRPC5 channel antagonist), significantly reduced nociceptive scores following cold water (0.1 ml) stimulation of the exposed dentin surface of the incisors compared to the scores of rats without local or systemic treatment. When we applied cold water stimulation to the exposed dentin surface of the lower first molar, nociceptive scores in the rats with systemic administration of A-317491, PANX, and GsMTx-4 were significantly reduced compared to those in the rats without systemic treatment. Dentin-exposed mice, with somatic odontoblast-specific depletion, also showed significant reduction in the nociceptive scores compared to those of Cre-mediated transgenic mice, which did not show any type of cell deletion, including odontoblasts. In the odontoblast-eliminated mice, P2X receptor-positive A-neurons were morphologically intact. These results indicate that neurotransmission between odontoblasts and neurons mediated by the Piezo1/TRPA1-pannexin-1-P2X receptor axis is necessary for the development of dentinal pain. In addition, odontoblasts are necessary for sensory transduction to generate dentinal sensitivity as mechanosensory receptor cells.
PubMed: 36589456
DOI: 10.3389/fphys.2022.891759 -
International Journal of Molecular... Feb 2022Leptin is a non-glycosylated 16 kDa protein synthesized mainly in adipose cells. The main function of leptin is to regulate energy homeostasis and weight control in a... (Review)
Review
Leptin is a non-glycosylated 16 kDa protein synthesized mainly in adipose cells. The main function of leptin is to regulate energy homeostasis and weight control in a central manner. There is increasing evidence that leptin also has systemic effects, acting as a link between innate and acquired immune responses. The expression of leptin and its receptor in human dental pulp and periradicular tissues have already been described, as well as several stimulatory effects of leptin protein expression in dental and periodontal tissues. The aim of this paper was to review and to compile the reported scientific literature on the role and effects of leptin in the dental pulp and periapical tissues. Twelve articles accomplished the inclusion criteria, and a comprehensive narrative review was carried out. Review of the available scientific literature concluded that leptin has the following effects on pulpal and periapical physiology: 1) Stimulates odontogenic differentiation of dental pulp stem cells (DPSCs), 2) Increases the expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1), odontoblastic proteins involved in odontoblastic differentiation and dentin mineralization, 3) Stimulates vascular endothelial growth factor (VEGF) expression in human dental pulp tissue and primary cultured cells of human dental pulp (hDPCs), 4) Stimulates angiogenesis in rat dental pulp cells, and 5) Induces the expression of interleucinas 6 and 8 in human periodontal ligament cells (hPDLCs). There is evidence which suggests that leptin is implicated in the dentin mineralization process and in pulpal and periapical inflammatory and reparative responses.
Topics: Animals; Cell Differentiation; Dental Pulp; Humans; Leptin; Odontogenesis; Periodontal Ligament
PubMed: 35216099
DOI: 10.3390/ijms23041984 -
Journal of Dental Research Jun 2024Located at the interface of the dentin-pulp complex, the odontoblasts are specialized cells responsible for dentin synthesis and nociceptive signal detection in response...
Located at the interface of the dentin-pulp complex, the odontoblasts are specialized cells responsible for dentin synthesis and nociceptive signal detection in response to external stimuli. Recent studies have shown that the mechanosensitive ion channel PIEZO1 is involved in bone formation and remodeling through the influx of calcium ions, and it is abundantly expressed in odontoblasts. However, the specific role of PIEZO1 in reactionary dentinogenesis and the underlying mechanisms remain elusive. In this study, we found intense PIEZO1 expression in the plasma membrane and cytoplasm of odontoblasts in healthy human third molars, mouse mandibular molars, and human odontoblast-like cells (hOBLCs). In hOBLCs, PIEZO1 positively regulated DSPP, DMP1, and COL1A1 expression through the Ca/PI3K-Akt/SEMA3A signaling pathway. In addition, exogenous SEMA3A supplementation effectively reversed reduced mineralization capacity in -knockdown hOBLCs. In vivo, Piezo1 expression peaked at day 7 and returned to baseline at day 21 in a wild-type mice dentin injury model, with Sema3a presenting a similar expression pattern. To investigate the specific role of PIEZO1 in odontoblast-mediated reactionary dentinogenesis, mice with a conditional knockout of in odontoblasts were generated, and no significant differences in teeth phenotypes were observed between the control and conditional knockout () mice. Nevertheless, mice exhibited reduced reactionary dentin formation and decreased Sema3a and Dsp positive staining after dentin injury, indicating impaired dental pulp repair by odontoblasts. In summary, these findings suggest that PIEZO1 enhances the mineralization capacity of hOBLCs in vitro via the Ca/PI3K-Akt/SEMA3A signaling pathway and contributes to reactionary dentinogenesis in vivo.
PubMed: 38910430
DOI: 10.1177/00220345241257866 -
European Cells & Materials Jul 2021Dentineogenesis starts on odontoblasts, which synthesise and secrete non-collagenous proteins (NCPs) and collagen. When dentine is injured, dental pulp... (Review)
Review
Dentineogenesis starts on odontoblasts, which synthesise and secrete non-collagenous proteins (NCPs) and collagen. When dentine is injured, dental pulp progenitors/mesenchymal stem cells (MSCs) can migrate to the injured area, differentiate into odontoblasts and facilitate formation of reactionary dentine. Dental pulp progenitor cell/MSC differentiation is controlled at given niches. Among dental NCPs, dentine sialophosphoprotein (DSPP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family, whose members share common biochemical characteristics such as an Arg-Gly-Asp (RGD) motif. DSPP expression is cell- and tissue-specific and highly seen in odontoblasts and dentine. DSPP mutations cause hereditary dentine diseases. DSPP is catalysed into dentine glycoprotein (DGP)/sialoprotein (DSP) and phosphoprotein (DPP) by proteolysis. DSP is further processed towards active molecules. DPP contains an RGD motif and abundant Ser-Asp/Asp-Ser repeat regions. DPP-RGD motif binds to integrin αVβ3 and activates intracellular signalling via mitogen-activated protein kinase (MAPK) and focal adhesion kinase (FAK)-ERK pathways. Unlike other SIBLING proteins, DPP lacks the RGD motif in some species. However, DPP Ser-Asp/Asp-Ser repeat regions bind to calcium-phosphate deposits and promote hydroxyapatite crystal growth and mineralisation via calmodulin-dependent protein kinase II (CaMKII) cascades. DSP lacks the RGD site but contains signal peptides. The tripeptides of the signal domains interact with cargo receptors within the endoplasmic reticulum that facilitate transport of DSPP from the endoplasmic reticulum to the extracellular matrix. Furthermore, the middle- and COOH-terminal regions of DSP bind to cellular membrane receptors, integrin β6 and occludin, inducing cell differentiation. The present review may shed light on DSPP roles during odontogenesis.
Topics: Cell Differentiation; Dental Pulp; Dentin; Extracellular Matrix Proteins; Odontoblasts; Phosphoproteins; Sialoglycoproteins
PubMed: 34275129
DOI: 10.22203/eCM.v042a04 -
ACS Biomaterials Science & Engineering Sep 2019Odontoblast processes, which grow inside dentin tubules, are critical parts of odontoblasts, and they play an important role in dentin hypersensitivity. However,...
Odontoblast processes, which grow inside dentin tubules, are critical parts of odontoblasts, and they play an important role in dentin hypersensitivity. However, modeling the growth of odontoblast processes is difficult, which hinders the study of dentin hypersensitivity and other dental diseases. To our knowledge, no technique has yet been developed to induce the growth of odontoblast processes from a cell body . In the current study, we fabricated a microfluidic chip via soft lithography. The microchannels on the chip can mimic the microstructures of dentin tubules, and the microchambers that connect to the microchannels can be used for odontoblast culture. We successfully induced the growth of odontoblast processes from cell bodies by using this chip. In addition, we designed chips with different microchannel sizes (i.e., 2, 4, 6, and 8 μm) to investigate the relationship between the growth of odontoblast processes and the geometric constraint imposed by microchannels. Experimental results show that the growth of odontoblast processes can be successfully induced by 2 μm channels. However, odontoblasts will migrate in the 4, 6, and 8 μm channels. This finding indicates that 2 μm is the appropriate size for inducing the growth of odontoblast processes . This value is consistent with the size of dentin tubules . The fabricated microfluidic chip can serve as a powerful tool for investigating the physiology and pathology of odontoblast processes in the future and developing treatment solutions for dental diseases, such as dentin hypersensitivity.
PubMed: 33448827
DOI: 10.1021/acsbiomaterials.9b00743 -
Journal of Dental Research May 2021The tooth is mainly composed of dentin and enamel. Identification of dentin-producing odontoblasts and enamel-producing ameloblasts using reporter techniques is useful...
The tooth is mainly composed of dentin and enamel. Identification of dentin-producing odontoblasts and enamel-producing ameloblasts using reporter techniques is useful to study tooth development and regeneration with tissue engineering. Ameloblasts express , and , whereas odontoblasts express () and (). Although there are several transgenic lines using promoter elements or bacterial artificial chromosomes (BACs) to label odontoblasts and ameloblasts, there is a possibility that the expression patterns vary from the endogenous genes. Here, we established 2 lines of mice where tdTomato was knocked into the second exon of X-chromosomal (), and green fluorescent protein (GFP) was knocked into the second exon of . tdTomato and GFP were highly expressed on secretory ameloblasts and secretory and fully differentiated odontoblasts, respectively. In addition, DSPP and AMELX were not produced in the dentin matrix and enamel matrix of and male mice (as representative of hemizygous male mice), respectively. Moreover, micro-computed tomography analysis of male mice revealed a notable reduction in enamel volume but increased dentin mineral density. mice had reduced dentin mineral density. To identify odontoblasts and ameloblasts from developing tooth, we examined the expression of mesenchymal cell surface molecules CD90, CD166 and epithelial cell surface molecules CD49f, Epcam1 with fluorescence on odontoblasts and ameloblasts in these mice. We found that GFP odontoblasts and tdTomato ameloblasts in tooth germ from 0.5-d-old mice and male mice were enriched in CD45/Ter119/Epcam1/CD90/Integrin α4cell fractions and CD45/Ter119/Epcam1/CD49f/CD147 cell fractions, respectively. By using antibodies against mesenchymal and epithelial cell surface molecules and fluorescence, we can easily distinguish odontoblasts from ameloblasts and isolate each cell for further studies. These mice would serve as useful models for tooth development and regeneration as well as provide concurrent observation for the differentiation processes of odontoblasts and ameloblasts in vivo and in vitro.
Topics: Ameloblasts; Animals; Cell Differentiation; Extracellular Matrix Proteins; Gene Knock-In Techniques; Male; Mice; Mice, Transgenic; Odontoblasts; Phosphoproteins; Sialoglycoproteins; X-Ray Microtomography
PubMed: 33289448
DOI: 10.1177/0022034520974576 -
Biomimetics (Basel, Switzerland) Oct 2023Assessing the biocompatibility of endodontic root-end filling materials through cell line responses is both essential and of utmost importance. This study aimed to the...
Assessing the biocompatibility of endodontic root-end filling materials through cell line responses is both essential and of utmost importance. This study aimed to the cytotoxicity of the type of cell death through apoptosis and autophagy, and odontoblast cell-like differentiation effects of MTA, zinc oxide-eugenol, and two experimental Portland cements modified with bismuth (Portland Bi) and barium (Portland Ba) on primary cell cultures. Material and methods: The cells corresponded to human periodontal ligament and gingival fibroblasts (HPLF, HGF), human pulp cells (HPC), and human squamous carcinoma cells from three different patients (HSC-2, -3, -4). The cements were inoculcated in different concentrations for cytotoxicity evaluation, DNA fragmentation in electrophoresis, apoptosis caspase activation, and autophagy antigen reaction, odontoblast-like cells were differentiated and tested for mineral deposition. The data were subject to a non-parametric test. Results: All cements caused a dose-dependent reduction in cell viability. Contact with zinc oxide-eugenol induced neither DNA fragmentation nor apoptotic caspase-3 activation and autophagy inhibitors (3-methyladenine, bafilomycin). Portland Bi accelerated significantly ( < 0.05) the differentiation of odontoblast-like cells. Within the limitation of this study, it was concluded that Portland cement with bismuth exhibits cytocompatibility and promotes odontoblast-like cell differentiation. This research contributes valuable insights into biocompatibility, suggesting its potential use in endodontic repair and biomimetic remineralization.
PubMed: 37999155
DOI: 10.3390/biomimetics8070514 -
Journal of Oral Biosciences Mar 2020The Bone Morphogenetic Proteins (BMPs) direct tooth development and still express in the adult tooth. We hypothesized that inhibition of BMP function would therefore...
OBJECTIVES
The Bone Morphogenetic Proteins (BMPs) direct tooth development and still express in the adult tooth. We hypothesized that inhibition of BMP function would therefore disrupt dentinogenesis by differentiated odontoblasts.
METHODS
We generated mice overexpressing the BMP-inhibitory protein Noggin in differentiated odontoblasts and osteocytes under control of a Dmp1 promoter-driven cre transgene. We compared the dentin phenotype in these mice with that in WT littermates and in mice with a Smad4 odontoblast/osteocyte knockout mediated by the same cre and therefore lacking all BMP and Tgfβ signaling in the same tissues.
RESULTS
Three-month-old first molars from both Noggin-expressing and Smad4-deleted mice showed decreased dentin volume with enlarged pulp cavities, and both displayed less organized and mineralized dentinal tubules compared to WT. The Smad4-ablated phenotype was more severe. While dentin sialophosphoprotein (DSPP) and bone sialoprotein (BSP) were decreased in the dentin of both lines, dentin matrix protein 1 (DMP1) was sharply increased in Noggin-expressing teeth.
CONCLUSIONS
The phenotypes we observed in Noggin-overexpressing and Smad4-conditional knockout teeth resemble the phenotype of Dentinogenesis Imperfecta (DGI) type III. Our results show that BMPs regulate post-natal dentinogenesis and that BMP-inhibitory proteins like Noggin play a role in that regulation. The increased severity of the Smad4 phenotype indicates that Tgfβ ligands, in addition to BMPs, play a crucial role in post-developmental dentinogenesis.
Topics: Animals; Carrier Proteins; Dentin; Dentinogenesis; Extracellular Matrix Proteins; Mice; Phosphoproteins; Sialoglycoproteins
PubMed: 31862386
DOI: 10.1016/j.job.2019.11.001 -
Tissue & Cell Dec 2022The calcium-binding protein S100A4 has been found in fibroblasts in many tissues. It has been well studied in the regulating process of inflammation and tumor...
INTRODUCTION
The calcium-binding protein S100A4 has been found in fibroblasts in many tissues. It has been well studied in the regulating process of inflammation and tumor metastasis. However, as a calcium regulating protein, its expression in the dental pulp has not yet been elucidated. The aim of this study was to investigate the in situ expression of S100A4 in dental pulp tissue.
METHODS
Five intact and three carious human teeth were collected and sectioned. Hematoxylin and eosin (HE) staining was used to locate areas reflecting the characteristics of the tooth. Based on this initial evaluation, the slides adjacent to these areas were immunohistochemically stained with S100A4 and dentin sialophosphoprotein (DSPP) antibodies.
RESULTS
In both the crown and root, S100A4 staining was observed in the odontoblast layer and in odontoblast-like cells, but not in other pulp cells. In contrast, DSPP was expressed in most cells of the dental pulp.
CONCLUSION
S100A4 is expressed in the odontoblast layer and in odontoblast-like cells in mature human pulp tissue. This protein can be used as a marker to differentiate these two kinds of cells from pulp cells.
Topics: Humans; Odontoblasts; Fibroblasts; Staining and Labeling; Antibodies; Calcium-Binding Proteins; S100 Calcium-Binding Protein A4
PubMed: 36327571
DOI: 10.1016/j.tice.2022.101959