Search

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine–phosphate–guanine (CpG) motifs (CpG-ODNs) are ligand molecules for Toll-like receptor 9 (TLR9), which is expressed by odontoblasts in vitro and dental pulp cells. This study determined the effects of CpG-ODNs on pulpal immunomodulatory response and repair following injury. Briefly, the upper right first molars of three-week-old mice were extracted, immersed in Type A (D35) or B (K3) CpG-ODN solutions (0.1 or 0.8 mM) for 30 min, and then replanted. Pulpal healing and immunomodulatory activity were assessed by hematoxylin–eosin and AZAN staining, as well as immunohistochemistry. One week following the operation, inflammatory reactions occurred in all of the experimental groups; however, re-revascularization and newly formed hard tissue deposition were observed in the pulp chamber of all groups at week 2. A positive trend in the expression of immune cell markers was observed toward the CpG-ODN groups at 0.1 mM. Our data suggest that synthetic CpG-ODN solutions at low concentrations may evoke a long-lasting macrophage–TLR9-mediated pro-inflammatory, rather than anti-inflammatory, response in the dental pulp to modulate the repair process and hard tissue formation. Further studies are needed to determine the effects of current immunomodulatory agents in vitro and in vivo and develop treatment strategies for dental tissue regeneration. [ABSTRACT FROM AUTHOR]

To investigate the transdentinal effects of surface reaction-type pre-reacted glass-ionomer (S-PRG) fillers on odontoblast-like cells. An eluate of S-PRG fillers was obtained by dissolving the particles in distilled water (1:1 m/v). Dentin discs with similar permeability were mounted into artificial pulp chambers and MDPC-23 cells were seeded on their pulpal surface. The occlusal surface was treated with (n = 10): ultrapure water (negative control – NC), hydrogen peroxide (positive control – PC), S-PRG eluate exposure for 1 min (S-PRG 1 min), or S-PRG filler eluate exposure for 30 min (S-PRG 30 min). After 24 h, cell viability (alamarBlue) and morphology (SEM) were evaluated. The extract obtained from transdentinal diffusion was applied to MDPC-23 pre-cultured in plates for another 24 h to evaluate viability (alamarBlue, 1, 3, and 7 days), gene expression of Col1a1 , Alpl , Dspp , and Dmp1 (RT-qPCR, 1 and 7 days), and mineralization (Alizarin Red, 7 days). Data were analyzed with ANOVA (α = 5 %). While S-PRG 1 min did not differ from NC, S-PRG 30 min reduced 17.9 % viability of cells from discs. S-PRG treatments resulted in low cell detaching from dentin, and the remaining cells exhibited typical morphology or minor cytoplasmic contraction. S-PRG 30 min slightly increased cell viability (6 %) 1 day after contact with the extract. S-PRG treatments upregulated the expression of the investigated genes, especially after 1 day. S-PRG 30 min stimulated mineralization activity by 39.7 %. S-PRG filler eluate does not cause transdentinal cytotoxicity on odontoblast-like cells, and long-term exposure can stimulate their dentinogenic-related mineralization activity. The transdentinal elution of ions from S-PRG fillers is not expected to be harmful to the dental pulp and may exert bioactive effects by inducing dentin matrix deposition through the metabolism of underlying odontoblasts. [ABSTRACT FROM AUTHOR]

Sacoglossa is a monophyletic group of small suctorial herbivorous Нeterobranchia. Due to the biology of feeding, most sacoglossans (except Cylindrobulla) have a distinctive feeding apparatus, which includes a sucking muscular organ as well as a sacoglossan-type radula with one tooth in a transverse row, which pierces algal cells. Although there is obvious interest in the radular morphology and developmental modes of Sacoglossa, as these characters may provide some insight into the evolutionary history and phylogenetic relationships of Sacoglossa, data on radula formation and morphogenesis in this group are sparse. This paper presents original data on the direct development, formation, and morphogenesis of the radula of Limapontia senestra (Quatrefages, 1844) from the White Sea. Based on the data obtained, we describe the development and formation of the radula. They fit into the general pattern of heterochrony in the radula anlage, characteristic of all gastropods. The small number of odontoblasts in the formation of the adult radula of L. senestra is a distinctive feature of the radula of Heterobranchia. [ABSTRACT FROM AUTHOR]

Objective: Despite that, the odontoblasts of the dental pulp are considered a terminally differentiated type of cell. We were interested in investigating if they express any embryonic, mesenchymal, or neural stem cell markers, along with other differentiation markers they were reported to express previously. Methods: An immunohistochemistry study was performed on wisdom teeth extracted from healthy donors aged between 17 and 19 for dental reasons. Nine markers were tested: c-Myc, SOX2, MCAM, CD73, NCAM1, STRO1, osteocalcin, S100, and Thy1. Results: Odontoblasts expressed the following markers: embryonic stem cell markers SOX2, c-Myc, mesenchymal stem cell marker MCAM, the neural differentiation marker S100, and the osteogenic differentiation marker osteocalcin. Odontoblasts did not express the following markers: mesenchymal stem cell markers CD73, STRO1, Thy1, and neural stem cell marker NCAM1. Conclusion: These findings suggest that odontoblasts’ expression of these stem cell markers may enable them to dedifferentiate under certain conditions. Further investigation is needed into whether dental materials could induce such dedifferentiation for functional dentin regeneration.

Objectives: The aim of this study was to investigate the molecular mechanism underlying odontoblast damage repair in dentin hypersensitivity (DH) and the role of Yes-associated protein (YAP) in this process. Methods: The DH model was constructed in Sprague-Dawley (SD) rats, and the in vivo expression of Piezo1, Integrin αvβ3, YAP, and dentin sialophosphoprotein (DSPP) was detected by immunohistochemistry. COMSOL Multiphysics software was used to simulate the dentinal tubule fluid flow velocity and corresponding fluid shear stress (FSS) on the odontoblast processes. MDPC-23 cells were cultured in vitro and loaded with a peristaltic pump for 1 hour at FSS values of 0.1, 0.3, 0.5, and 0.7 dyne/cm2. The expression of Piezo1, Integrin αvβ3, and YAP was detected by immunofluorescence. Verteporfin (a YAP-specific inhibitor) was utilised to confirm the effect of YAP on the expression of dentineogenesis-related protein under FSS. Results: The level and duration of external mechanical stimuli have an effect on the functional expression of odontoblasts. In DH, the harder the food that is chewed, the faster the flow of the dentinal tubule fluid and the greater the FSS on the odontoblast processes. The expression of Piezo1, Integrin αvβ3, and YAP can be promoted when the FSS is less than 0.3 dyne/cm2. After YAP inhibition, the DSPP protein expression level was reduced at 0.3 dyne/cm2 FSS. Conclusions: These results suggest that appropriate FSS can enhance the expression of odontoblast-related factors in odontoblasts via the Piezo1–Integrin αvβ3–YAP mechanotransduction pathway and the YAP appears to play an essential role in the response of odontoblasts to external mechanical stimuli.

Introduction: Recent advancements in dental materials have led to the development of various restorative cements. The amalgamation of various biomimetic cements becomes necessary to improve the physico-chemical properties of the existing cements. Mineral Trioxide Aggregate (MTA) Plus and Chitosan are two such materials which are combined and chosen to check their effects on dental stem cell adhesion and proliferation. Aim: To compare and evaluate the cell adhesion and proliferation of human dental pulp stem cells on MTA Plus, MTA Plus-Chitosan Conjugate, and Chitosan by directly culturing the cells on the materials. Materials and Methods: This in-vitro study was conducted at the Central Research Laboratory in Department of Conservative Dentistry and Endodontics, S.D.M College of Dental Sciences, Dharwad, Karnataka, India over a period of 1.5 years from the month of June 2020 to December 2021. The samples were divided into four groups: Group A-MTA Plus mixed with the proprietary gel (9 specimens), Group B-MTA Plus mixed with 2% Chitosan gel (9 specimens), Group C-2% Chitosan Gel (9 specimens), and Group D-Control Group (Growth Medium). Non carious extracted permanent human premolars for orthodontic purposes were collected and sectioned for the extraction of pulp tissue. The tissue was placed in a basic medium Dulbecco modified essential medium-High Glucose (DMEM-HG). Dental Pulp Stem Cells were subjected to differentiation using differentiation media (Hi-Media) containing Dexamethasone. Cell proliferation and viability were checked using trypan blue, and cell adhesion was checked using crystal violet under an inverted light microscope. To evaluate if there were any changes in the proliferation of viable cells at 24 hours, 48 hours, and 72 hours among different materials, the Wilcoxon’s Signed-rank Test was used. Statistical significance was set at p≤0.05. Results: The cells were able to adhere to the biomaterial and showed a spindle-shaped morphology, which was observed under the inverted light microscope. ALP activity was measured using a photometer after cell differentiation. MTA Plus-Chitosan conjugate showed a 94% increase in cell proliferation after 48 hours (n=86/91). MTA Plus showed a 91% increase in cell proliferation after 48 hours (n=109/120). Conclusion: Chitosan can be used as a vehicle with MTA since their conjugate showed greater proliferation activity after stem cell culture.

Epigenetic modulation, including histone modification, alters gene expression and controls cell fate. Histone deacetylases (HDACs) are identified as important regulators of dental pulp cell (DPC) mineralisation processes. Currently, there is a paucity of information regarding the nature of histone modification and HDAC expression in the dentine–pulp complex during dentinogenesis. The aim of this study was to investigate post-translational histone modulation and HDAC expression during DPC mineralisation and the expression of Class I/II HDACs during tooth development and in adult teeth. HDAC expression (isoforms −1 to −6) was analysed in mineralising primary rat DPCs using qRT-PCR and Western blot with mass spectrometry being used to analyse post-translational histone modifications. Maxillary molar teeth from postnatal and adult rats were analysed using immunohistochemical (IHC) staining for HDACs (1–6). HDAC-1, -2, and -4 protein expression increased until days 7 and 11, but decreased at days 14 and 21, while other HDAC expression increased continuously for 21 days. The Class II mineralisation-associated HDAC-4 was strongly expressed in postnatal sample odontoblasts and DPCs, but weakly in adult teeth, while other Class II HDACs (-5, -6) were relatively strongly expressed in postnatal DPCs and adult odontoblasts. Among Class I HDACs, HDAC-1 showed high expression in postnatal teeth, notably in ameloblasts and odontoblasts. HDAC-2 and -3 had extremely low expression in the rat dentine–pulp complex. Significant increases in acetylation were noted during DPC mineralisation processes, while trimethylation H3K9 and H3K27 marks decreased, and the HDAC-inhibitor suberoylanilide hydroxamic acid (SAHA) enhanced H3K27me3. These results highlight a dynamic alteration in histone acetylation during mineralisation and indicate the relevance of Class II HDAC expression in tooth development and regenerative processes. [ABSTRACT FROM AUTHOR]

Despite that, the odontoblasts of the dental pulp are considered a terminally differentiated type of cell. We were interested in investigating if they express any embryonic, mesenchymal, or neural stem cell markers, along with other differentiation markers they were reported to express previously. Methods : An immunohistochemistry study was performed on wisdom teeth extracted from healthy donors aged between 17 and 19 for dental reasons. Nine markers were tested: c-Myc, SOX2, MCAM, CD73, NCAM1, STRO1, osteocalcin, S100, and Thy1. Results : Odontoblasts expressed the following markers: embryonic stem cell markers SOX2, c-Myc, mesenchymal stem cell marker MCAM, the neural differentiation marker S100, and the osteogenic differentiation marker osteocalcin. Odontoblasts did not express the following markers: mesenchymal stem cell markers CD73, STRO1, Thy1, and neural stem cell marker NCAM1. Conclusion : These findings suggest that odontoblasts' expression of these stem cell markers may enable them to dedifferentiate under certain conditions. Further investigation is needed into whether dental materials could induce such dedifferentiation for functional dentin regeneration. [ABSTRACT FROM AUTHOR]

Aim: The aim of the study was to evaluate the ability of cultivated odontoblast to form dentin-like tissue using fibroblast growth factor (FGF) and insulin-like growth factor (IGF). Materials and Methods: Dental pulp stem cells (DPSCs) were extracted from 10 human teeth. They were isolated and cultivated in vitro with the use of stem cell markers. The human DPSCs were characterized for trilineage differentiation. They were then differentiated into odontoblasts. The ability of cultivated odontoblasts to form dentin-like tissue was evaluated using FGF and IGF. Results: IGF showed superior ability to form dentin-like tissue as compared to FGF. The addition of FGF showed no significant difference in the formation of dentin-like tissue. A combination of FGF and IGF in odontoblast showed an enhanced ability to form dentin-like tissue. Conclusion: The use of growth factors IGF and FGF with dental stem cells showed a greater potential to form dentin-like tissue. This can profoundly alter the paradigms of conservative vital pulp therapy, which may eventually make it possible to treat dental diseases by regeneration of lost dentine. [ABSTRACT FROM AUTHOR]

Introduction: Recent advancements in dental materials have led to the development of various restorative cements. The amalgamation of various biomimetic cements becomes necessary to improve the physico-chemical properties of the existing cements. Mineral Trioxide Aggregate (MTA) Plus and Chitosan are two such materials which are combined and chosen to check their effects on dental stem cell adhesion and proliferation. Aim: To compare and evaluate the cell adhesion and proliferation of human dental pulp stem cells on MTA Plus, MTA Plus-Chitosan Conjugate, and Chitosan by directly culturing the cells on the materials. Materials and Methods: This in-vitro study was conducted at the Central Research Laboratory in Department of Conservative Dentistry and Endodontics, S.D.M College of Dental Sciences, Dharwad, Karnataka, India over a period of 1.5 years from the month of June 2020 to December 2021. The samples were divided into four groups: Group A-MTA Plus mixed with the proprietary gel (9 specimens), Group B-MTA Plus mixed with 2% Chitosan gel (9 specimens), Group C-2% Chitosan Gel (9 specimens), and Group D-Control Group (Growth Medium). Non carious extracted permanent human premolars for orthodontic purposes were collected and sectioned for the extraction of pulp tissue. The tissue was placed in a basic medium Dulbecco modified essential medium-High Glucose (DMEM-HG). Dental Pulp Stem Cells were subjected to differentiation using differentiation media (Hi-Media) containing Dexamethasone. Cell proliferation and viability were checked using trypan blue, and cell adhesion was checked using crystal violet under an inverted light microscope. To evaluate if there were any changes in the proliferation of viable cells at 24 hours, 48 hours, and 72 hours among different materials, the Wilcoxon's Signed-rank Test was used. Statistical significance was set at p=0.05. Results: The cells were able to adhere to the biomaterial and showed a spindle-shaped morphology, which was observed under the inverted light microscope. ALP activity was measured using a photometer after cell differentiation. MTA Plus-Chitosan conjugate showed a 94% increase in cell proliferation after 48 hours (n=86/91). MTA Plus showed a 91% increase in cell proliferation after 48 hours (n=109/120). Conclusion: Chitosan can be used as a vehicle with MTA since their conjugate showed greater proliferation activity after stem cell culture. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this research was to investigate the functions of Piezo channels in dentin defect, including mechanical signalling and odontoblast responses. Methods: Rat dentin-defect models were constructed, and spatiotemporal expression of Piezo proteins was detected in the pulpo-dentinal complex. Real-time polymerase chain reaction (rtPCR) was used to investigate the functional expression pattern of Piezo channels in odontoblasts. Moreover, RNA interference technology was employed to uncover the underlying mechanisms of the Piezo-driven inflammatory response and repair under fluid shear stress (FSS) conditions in vitro. Results: Piezo1 and Piezo2 were found to be widely expressed in the odontoblast layer and dental pulp in the rat dentin-defect model during the end stage of reparative dentin formation. The expression levels of the Piezo1 and Piezo2 genes in MDPC-23 cells were high in the initial stage under FSS loading and then decreased over time. Moreover, the expression trends of inflammatory, odontogenic, and mineralisation genes were generally contrary to those of Piezo1 and Piezo2 over time. After silencing of Piezo1/Piezo2, FSS stimulation resulted in significantly higher expression of inflammatory, odontogenesis, and mineralisation genes in MDPC-23 cells. Finally, the expression of genes involved in the integrin β1/ERK1 and Wnt5b/β-catenin signalling pathways was changed in response to RNA silencing of Piezo1 and Piezo2. Conclusions: Piezo1 and Piezo2 may be involved in regulating the expression of inflammatory and odontogenic genes in odontoblasts stimulated by FSS.

Immunoglobulin (Ig)A nephropathy has been associated with oral infections such as periodontitis, but its pathogenesis is not fully understood; no treatments exist. This study analyzes the influence of IgA nephropathy, an autoimmune disease, on the pathogenesis of pulpitis and apical periodontitis. Two groups of mice were used in pulp infection experiments: high serum IgA nephropathy model mice (HIGA) and control mice (BALB/c). Histologic analyses of the pulp and apical periodontal tissues were performed on days 3, 5, 7, 14, and 28 following oral bacterial infection. The dynamics of odontoblasts, apoptotic cells, and IgA expression were analyzed using anti-Nestin, TUNEL, and anti-IgA staining, respectively. Inflammatory cells infiltrated the exposed pulp at day three in both groups and by 14 days, these cells had infiltrated from the pulp to the apical periodontal tissue. The area of necrotic pulp tissue increased significantly in the control group at seven days. Odontoblasts decreased from day three onwards and disappeared by 28 days in both groups. The number of apoptotic cells in the pulp and apical periodontal tissues was significantly higher in the experimental group at day 28. The experimental group exhibited a significant increase in IgA production in the pulp after 14 days. Bone resorption in the apical periodontal tissue was significantly decreased in the experimental group at day 28. The results of this study suggest that IgA nephropathy may modulate the inflammatory response and sustain long-term biological defense responses in pulpitis and apical periodontitis in HIGA mice. [ABSTRACT FROM AUTHOR]

Regenerative endodontic procedures (REPs) are promising for dental pulp tissue regeneration; however, their application in permanent teeth remains challenging. We assessed the potential combination of an REP and local dental pulp cell (DPC) transplantation in the mature molars of C57BL/6 mice with (REP + DPC group) or without (REP group) transplantation of DPCs from green fluorescent protein (GFP) transgenic mice. After 4 weeks, the regenerated tissue was evaluated by micro-computed tomography and histological analyses to detect odontoblasts, vasculogenesis, and neurogenesis. DPCs were assessed for mesenchymal and pluripotency markers. Four weeks after the REP, the molars showed no signs of periapical lesions, and both the REP and REP + DPC groups exhibited a pulp-like tissue composed of a cellular matrix with vessels surrounded by an eosin-stained acellular matrix that resembled hard tissue. However, the REP + DPC group had a broader cellular matrix and uniquely contained odontoblast-like cells co-expressing GFP. Vasculogenesis and neurogenesis were detected in both groups, with the former being more prominent in the REP + DPC group. Overall, the REP was achieved in mature mouse molars and DPC transplantation improved the outcomes by inducing the formation of odontoblast-like cells and greater vasculogenesis. [ABSTRACT FROM AUTHOR]

Mouse and human genetic studies indicate key roles of the Wnt10a ligand in odontogenesis. Previous studies have identified effectors and regulators of the Wnt signaling pathway actively expressed during key stages of tooth morphogenesis. However, limitations in multiplexing and spatial resolution hindered a more comprehensive analysis of these signaling molecules. Here, profiling of transcriptomes using fluorescent multiplex in situ hybridization and single-cell RNA-sequencing (scRNA-seq) provide robust insight into the synchronized expression patterns of Wnt10a, Dkk1, and Sost simultaneously during tooth development. First, we identified Wnt10a transcripts restricted to the epithelium at the stage of tooth bud morphogenesis, contrasting that of Sost and Dkk1 localization to the dental mesenchyme. By embryonic day 15.5 (E15.5), a marked shift of Wnt10a expression from dental epithelium to mesenchyme was noted, while Sost and Dkk1 expression remained enriched in the mesenchyme. By postnatal day 0 (P0), co-localization patterns of Wnt10a, Dkk1, and Sost were observed in both terminally differentiating and secreting odontoblasts of molars and incisors. Interestingly, Wnt10a exhibited robust expression in fully differentiated ameloblasts at the developing cusp tip of both molars and incisors, an observation not previously noted in prior studies. At P7 and 14, after the mineralization of dentin and enamel, Wnt10a expression was limited to odontoblasts. Meanwhile, Wnt modulators showed reduced or absent signals in molars. In contrast, strong signals persisted in ameloblasts (for Wnt10a) and odontoblasts (for Wnt10a, Sost, and Dkk1) towards the proximal end of incisors, near the cervical loop. Our scRNA-seq analysis used CellChat to further contextualize Wnt pathway-mediated communication between cells by examining ligand-receptor interactions among different clusters. The colocalization pattern of Wnt10a, Dkk1, and Sost in both terminally differentiating and secreting odontoblasts of molars and incisors potentially signifies the crucial ligand-modulator interaction along the gradient of cytodifferentiation starting from each cusp tip towards the apical region. These data provide cell type-specific insight into the role of Wnt ligands and mediators during epithelial-mesenchymal interactions in odontogenesis. [ABSTRACT FROM AUTHOR]

This in vitro experimental investigation aimed to evaluate the impact of the combined application of a nanofiber scaffold (NS), a polymeric catalyst primer (PCP) containing 10 mg/mL of heme peroxidase enzyme, and violet LED (LEDv) on the esthetic efficacy (EE), trans-amelodentinal cytotoxicity (TC), and procedural duration of conventional in-office bleaching therapy. To achieve this, 96 standardized enamel/dentin discs were individually placed in artificial pulp chambers. A 35% hydrogen peroxide (H2O2) bleaching gel was administered for 45, 30, or 15 min to the enamel, either previously coated with NS + PCP or left uncoated, followed by irradiation with LEDv for 15 min or no irradiation. The established groups were as follows: G1, negative control (no treatment); G2, 35% H2O2/45 min; G3, NS + PCP + LEDv; G4, NS + PCP + 35%H2O2/45 min + LEDv; G5, NS + PCP + 35%H2O2/30 min + LEDv; and G6, NS + PCP + 35%H2O2/15 min + LEDv. Extracts (culture medium + gel components diffused through the discs) were collected and applied to odontoblast-like MDPC-23 cells. EE (ΔE00 and ΔWI) and TC were assessed using ANOVA/Tukey analysis (p < 0.05). The EE analysis revealed no statistical differences between G6 and G2 (p > 0.05). Cells in G6 exhibited higher viability and lower oxidative stress compared to other bleached groups (p < 0.05). In conclusion, employing NS + PCP + LEDv to catalyze a 35%H2O2 bleaching gel applied for 15 min to the enamel resulted in successful esthetic improvements and reduced the cytotoxicity commonly linked with traditional in-office bleaching procedures. [ABSTRACT FROM AUTHOR]

Perfect intercellular junctions are key for odontoblast barrier function. However, whether Partitioning defective-3 (Par3) is expressed in odontoblasts and its potential effects on odontoblast junctions are unknown. Herein, we investigated the effect of Par3 on cellular junctions and the biological behavior of odontoblast-lineage cells (OLCs). Whole-transcriptome sequencing was used to analyze the effects of Par3 on OLCs and the underlying molecular mechanism. Par3 was detected under physiological and inflammatory conditions in OLCs. To investigate the regulatory effect of Par3 on junctions between mouse OLCs, the effects of Par3 downregulation on the proliferation, migration, cycle and apoptosis of OLCs were detected by 5-ethyl-2′-deoxyuridine (EdU) and Transwell assays and flow cytometry. Western blotting and alizarin red S and alkaline phosphatase (ALP) staining were used to observe the effect of Par3 downregulation on OLC mineralization. Whole-transcriptome sequencing was used to investigate the biological role of Par3 in OLCs and potential molecular mechanisms. Par3 was located along the odontoblast layer in the rat pulp tissue and in the cytoplasm of OLCs. Par3 expression was downregulated under inflammatory conditions. The OLC junctions were discontinuous, and total Zona occluden-1 (ZO-1) expression and expression of ZO-1 at the membrane in OLCs were reduced after Par3 silencing (P < 0.05). Expression of a junction-related protein (ZO-1) was downregulated after the downregulation of Par3 (P < 0.05), and ZO-1 moved from the cell membrane to the cytoplasm. OLC proliferation and migration were enhanced, but apoptosis and mineralization were inhibited in shPar3-transfected cells (P < 0.05). Sequencing identified 2996 differentially expressed genes (DEGs), which were mainly enriched in the response to stimuli and binding. Downregulation of Par3 could overactivate the PI3k-AKT pathway by promoting AKT phosphorylation (P < 0.05). Downregulation of Par3 may disrupt junctions between OLCs by affecting ZO-1 expression and distribution and promote OLC proliferation and migration but inhibit OLC mineralization. Par3 may interact with 14-3-3 proteins for PI3K-AKT pathway activation to affect OLC junctions and function. [ABSTRACT FROM AUTHOR]

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine–phosphate–guanine (CpG) motifs (CpG-ODNs) are ligand molecules for Toll-like receptor 9 (TLR9), which is expressed by odontoblasts in vitro and dental pulp cells. This study determined the effects of CpG-ODNs on pulpal immunomodulatory response and repair following injury. Briefly, the upper right first molars of three-week-old mice were extracted, immersed in Type A (D35) or B (K3) CpG-ODN solutions (0.1 or 0.8 mM) for 30 min, and then replanted. Pulpal healing and immunomodulatory activity were assessed by hematoxylin–eosin and AZAN staining, as well as immunohistochemistry. One week following the operation, inflammatory reactions occurred in all of the experimental groups; however, re-revascularization and newly formed hard tissue deposition were observed in the pulp chamber of all groups at week 2. A positive trend in the expression of immune cell markers was observed toward the CpG-ODN groups at 0.1 mM. Our data suggest that synthetic CpG-ODN solutions at low concentrations may evoke a long-lasting macrophage–TLR9-mediated pro-inflammatory, rather than anti-inflammatory, response in the dental pulp to modulate the repair process and hard tissue formation. Further studies are needed to determine the effects of current immunomodulatory agents in vitro and in vivo and develop treatment strategies for dental tissue regeneration.

Background. Dental papilla cells (DPCs) are one of the key stem cells for tooth development, eventually forming dentin and pulp. Previous studies have reported that PER2 is expressed in a 24-hour oscillatory pattern in DPCs in vitro. In vivo, PER2 is highly expressed in odontoblasts (which are differentiated from DPCs). However, whether PER2 modulates the odontogenic differentiation of DPCs is uncertain. This research was to identify the function of PER2 in the odontogenic differentiation of DPCs and preliminarily explore its mechanisms. Methods. We monitored the expression of PER2 in DPCs differentiated in vivo. We used PER2 overexpression and knockdown studies to assess the role of PER2 in DPC differentiation and performed intracellular ATP content and reactive oxygen species (ROS) assays to further investigate the mechanism. Results. PER2 expression was considerably elevated throughout the odontoblastic differentiation of DPCs in vivo. Overexpressing Per2 boosted levels of odontogenic differentiation markers, such as dentin sialophosphoprotein (Dspp), dentin matrix protein 1 (Dmp1), and alkaline phosphatase (Alp), and enhanced mineralized nodule formation in DPCs. Conversely, the downregulation of Per2 inhibited the differentiation of DPCs. Additionally, downregulating Per2 further affected intracellular ATP content and ROS levels during DPC differentiation. Conclusion. Overall, we demonstrated that PER2 positively regulates the odontogenic differentiation of DPCs, and the mechanism may be related to mitochondrial function as shown by intracellular ATP content and ROS levels. [ABSTRACT FROM AUTHOR]

The dental pulp plays a crucial role in the long-term maintenance of tooth function. The progress of endodontic treatment and pulp tissue regeneration engineering has made pulp-regeneration therapy promising in clinical practice. However, the mechanisms of pulp regeneration and the role of dental stem cells in development and regeneration have not been fully elucidated. Bridging the gaps between clinical operation and basic research is urgently needed. With the application of single-cell sequencing technology in dental research, the landscapes of human dental pulp cells have begun being outlined. However, the specific cellular heterogeneity of dental pulp cells, especially that of dental stem cells, at different spatial and temporal levels, is still unclear. In this study, we used single-cell RNA sequencing analysis of pulp samples at four different developmental stages and combined the findings with immunohistochemical staining to explore the development of dental pulp and stem cells. The results revealed temporal changes in the proportion of pulp cells during development. For example, mononuclear phagocytes accounted for a higher proportion in early samples. Odontoblasts identified by DMP1 had a higher expression of ion channel-related and neurodevelopment-related genes. Subpopulations were identified in fibroblasts, odontoblasts, and mesenchymal stem cells. We identified a subclass of odontoblasts that expresses DGKI and RRBP1 present in early developmental samples. Population of earlier mesenchymal stem cells expressed the SEPTIN gene, which may have greater proliferative and differentiation potential. Furthermore, dental pulp stem cells can differentiate into two directions: mineralization and myogenesis. In summary, the specific cellular heterogeneity of dental pulp cells was revealed at different spatial and temporal levels. These findings may shed light on the mechanism of tooth development. The gene expression profile of developing pulp cells may help to select cells for regenerative engineering and improve the success of dental pulp regeneration. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is an autoimmune disease that involves joint inflammation. Although periodontal disease reportedly contributes to RA onset, the associations of RA with pulpitis and apical periodontitis have not been described. The purpose of this study was to examine the effects of immune response disruption of RA for pulpitis and apical periodontitis with SKG mice. SKG and BALB/c (control) mice were used to establish models of pulp infection. Histologic studies of pulp and apical periodontal tissue were performed at 3, 5, 7, 14, and 28 days; odontoblast dynamics were analyzed by antinestin staining, and apoptotic cells were examined by TdT-mediated digoxygenin (biotin)-dUTP nick end labeling staining. Inflammatory cell infiltration into the exposed pulp was observed at 3 days in the SKG and control group groups; the infiltration extended to the apical pulp area at 14 days after surgery. Inflammatory cell infiltration and bone resorption in the apical pulp area were observed from 14–28 days in the SKG and control groups; there were significant increases in inflammatory cell infiltration and bone resorption in the control group at 28 days. The numbers of apoptotic cells in pulp and apical periodontal tissue were higher in the SKG group than in the control group at 14 and 28 days. The number of odontoblasts decreased in the SKG and control groups until 14 days and then disappeared in the SKG and control groups at 28 days. This study suggested that immune response disruption in RA is involved in prolonging the inflammatory state of pulpitis and apical periodontitis. [ABSTRACT FROM AUTHOR]

Objective(s): The purpose of this study was to assess the effects of exosomes derived from dental pulp mesenchymal stem cells (DPSCs) on the viability of odontoblast cells. Methods: Exosomes were extracted from human DPSC cells via ultracentrifugation. Scanning electron microscopy and western blot analysis for CD9 and CD81 demonstrated characteristics of exosomes. Developing odontoblasts were exposed to increasing concentrations of exosomes (10-400 ng/ml) over time periods of 24, 48, 72, and 96 hours. Cell viability was evaluated using MTT assays. In addition, real-time PCR was used to assess expression levels of Wnt, betacatenin, PI3K, Akt, Bcl-2, and Bax in cells after 24-96 hours of exposure to 100 and 400 ng/ml exosomes. Results: Odontoblast viability was found to be enhanced by DPSC-derived exosomes, with greater impacts at higher concentrations and time periods of 72-96 hours. Expression of PI3K, AKT, Wnt, beta-catenin, and Bcl-2 were notably upregulated in odontoblasts following 96 hours of DPSC-exosome treatment. Conversely, Bax expression was significantly downregulated. These findings suggest DPSC-exosomes promote odontoblast survival through modulated expression of genes involved in key survival pathways over extended exposure periods. Conclusions: Our findings demonstrated that exosomes derived from DPSC cells possess innate nanoparticle properties, leading to enhanced survival of odontoblast cells. This effect is achieved through the activation of the Wnt/betacatenin and P3K/AKT signaling pathways, ultimately resulting in an increased Bcl2/Bax ratio. [ABSTRACT FROM AUTHOR]

Mouse and human genetic studies indicate key roles of the Wnt10a ligand in odontogenesis. Previous studies have identified effectors and regulators of the Wnt signaling pathway actively expressed during key stages of tooth morphogenesis. However, limitations in multiplexing and spatial resolution hindered a more comprehensive analysis of these signaling molecules. Here, profiling of transcriptomes using fluorescent multiplex in situ hybridization and single-cell RNA-sequencing (scRNA-seq) provide robust insight into the synchronized expression patterns of Wnt10a, Dkk1, and Sost simultaneously during tooth development. First, we identified Wnt10a transcripts restricted to the epithelium at the stage of tooth bud morphogenesis, contrasting that of Sost and Dkk1 localization to the dental mesenchyme. By embryonic day 15.5 (E15.5), a marked shift of Wnt10a expression from dental epithelium to mesenchyme was noted, while Sost and Dkk1 expression remained enriched in the mesenchyme. By postnatal day 0 (P0), co-localization patterns of Wnt10a, Dkk1, and Sost were observed in both terminally differentiating and secreting odontoblasts of molars and incisors. Interestingly, Wnt10a exhibited robust expression in fully differentiated ameloblasts at the developing cusp tip of both molars and incisors, an observation not previously noted in prior studies. At P7 and 14, after the mineralization of dentin and enamel, Wnt10a expression was limited to odontoblasts. Meanwhile, Wnt modulators showed reduced or absent signals in molars. In contrast, strong signals persisted in ameloblasts (for Wnt10a) and odontoblasts (for Wnt10a, Sost, and Dkk1) towards the proximal end of incisors, near the cervical loop. Our scRNA-seq analysis used CellChat to further contextualize Wnt pathway-mediated communication between cells by examining ligand-receptor interactions among different clusters. The co-localization pattern of Wnt10a, Dkk1, and Sost in both terminally differentiating and secreting odontoblasts of molars and incisors potentially signifies the crucial ligand-modulator interaction along the gradient of cytodifferentiation starting from each cusp tip towards the apical region. These data provide cell type-specific insight into the role of Wnt ligands and mediators during epithelial-mesenchymal interactions in odontogenesis.

Abstract 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+; TSC1f/f, 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.

Epigenetic modulation, including histone modification, alters gene expression and controls cell fate. Histone deacetylases (HDACs) are identified as important regulators of dental pulp cell (DPC) mineralisation processes. Currently, there is a paucity of information regarding the nature of histone modification and HDAC expression in the dentine–pulp complex during dentinogenesis. The aim of this study was to investigate post-translational histone modulation and HDAC expression during DPC mineralisation and the expression of Class I/II HDACs during tooth development and in adult teeth. HDAC expression (isoforms −1 to −6) was analysed in mineralising primary rat DPCs using qRT-PCR and Western blot with mass spectrometry being used to analyse post-translational histone modifications. Maxillary molar teeth from postnatal and adult rats were analysed using immunohistochemical (IHC) staining for HDACs (1–6). HDAC-1, -2, and -4 protein expression increased until days 7 and 11, but decreased at days 14 and 21, while other HDAC expression increased continuously for 21 days. The Class II mineralisation-associated HDAC-4 was strongly expressed in postnatal sample odontoblasts and DPCs, but weakly in adult teeth, while other Class II HDACs (-5, -6) were relatively strongly expressed in postnatal DPCs and adult odontoblasts. Among Class I HDACs, HDAC-1 showed high expression in postnatal teeth, notably in ameloblasts and odontoblasts. HDAC-2 and -3 had extremely low expression in the rat dentine–pulp complex. Significant increases in acetylation were noted during DPC mineralisation processes, while trimethylation H3K9 and H3K27 marks decreased, and the HDAC-inhibitor suberoylanilide hydroxamic acid (SAHA) enhanced H3K27me3. These results highlight a dynamic alteration in histone acetylation during mineralisation and indicate the relevance of Class II HDAC expression in tooth development and regenerative processes.

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

The use of stem cells for tissue regeneration is a prominent trend in regenerative medicine and tissue engineering. In particular, dental pulp stem cells (DPSCs) have garnered considerable attention. When exposed to specific conditions, DPSCs have the ability to differentiate into osteoblasts and odontoblasts. Scaffolds are critical for cell differentiation because they replicate the 3D microenvironment of the niche and enhance cell adhesion, migration, and differentiation. The purpose of this study is to present the biological responses of human DPSCs to a purified 3D chitin scaffold derived from the marine demosponge Aplysina fistularis and modified with hydroxyapatite (HAp). Responses examined included proliferation, adhesion, and differentiation. The control culture consisted of the human osteoblast cell line, hFOB 1.19. Electron microscopy was used to examine the ultrastructure of the cells (transmission electron microscopy) and the surface of the scaffold (scanning electron microscopy). Cell adhesion to the scaffolds was determined by neutral red and crystal violet staining methods. An alkaline phosphatase (ALP) assay was used for assessing osteoblast/odontoblast differentiation. We evaluated the expression of osteogenic marker genes by performing ddPCR for ALP, RUNX2, and SPP1 mRNA expression levels. The results show that the chitin biomaterial provides a favorable environment for DPSC and hFOB 1.19 cell adhesion and supports both cell proliferation and differentiation. The chitin scaffold, especially with HAp modification, isolated from A. fistularis can make a significant contribution to tissue engineering and regenerative medicine.

Mitochondrial transfer is emerging as a promising therapeutic strategy for tissue repair, but whether it protects against pulpitis remains unclear. Here, we show that hyperactivated nucleotide‐binding domain and leucine‐rich repeat protein3 (NLRP3) inflammasomes with pyroptotic cell death was present in pulpitis tissues, especially in the odontoblast layer, and mitochondrial oxidative stress (OS) was involved in driving this NLRP3 inflammasome‐induced pathology. Using bone marrow mesenchymal stem cells (BMSCs) as mitochondrial donor cells, we demonstrated that BMSCs could donate their mitochondria to odontoblasts via tunnelling nanotubes (TNTs) and, thus, reduce mitochondrial OS and the consequent NLRP3 inflammasome‐induced pyroptosis in odontoblasts. These protective effects of BMSCs were mostly blocked by inhibitors of the mitochondrial function or TNT formation. In terms of the mechanism of action, TNF‐α secreted from pyroptotic odontoblasts activates NF‐κB signalling in BMSCs via the paracrine pathway, thereby promoting the TNT formation in BMSCs and enhancing mitochondrial transfer efficiency. Inhibitions of NF‐κB signalling and TNF‐α secretion in BMSCs suppressed their mitochondrial donation capacity and TNT formation. Collectively, these findings demonstrated that TNT‐mediated mitochondrial transfer is a potential protective mechanism of BMSCs under stress conditions, suggesting a new therapeutic strategy of mitochondrial transfer for dental pulp repair. [ABSTRACT FROM AUTHOR]

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+; TSC1f/f, 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. [ABSTRACT FROM AUTHOR]

CPNE1 is a calcium‐dependent, phospholipid‐binding protein that is ubiquitously expressed in various tissues and organs. This study investigates the expression and localization of CPNE1 in tooth germ development and the role of CPNE1 in odontoblastic differentiation. In rat tooth germs, CPNE1 is expressed in the odontoblasts and ameloblasts since the late bell stage. The depletion of CPNE1 in the stem cells from apical papilla (SCAPs) clearly inhibits the expression of odontoblastic‐related genes and the formation of mineralized nodules during differentiation, while CPNE1 overexpression promotes this process. In addition, CPNE1 overexpression increases AKT phosphorylation during the odontoblastic differentiation of SCAPs. Furthermore, treatment with AKT inhibitor (MK2206) reduces the expression of odontoblastic‐related genes in CPNE1 over‐expressed SCAPs, and Alizarin Red staining shows reduced mineralization. These results suggest that CPNE1 plays a role in the tooth germ development as well as the odontblastic differentiation of SCAPs in vitro that is related to the AKT signaling pathway. [ABSTRACT FROM AUTHOR]

Introduction: Although there are many desensitizing medications on the market with promising results, their therapeutic advantages are sometimes fleeting or diminished over time since they might be gradually destroyed by consistent tooth brushing or ingestion of acidic beverages. Aim: In this scanning electron microscope study, an initial effort was made to evaluate and compare the dentinal changes that occurred after healthy fluorosed and nonfluorosed teeth were subjected to the application of 1% and 10% nano-hydroxyapatite (n-HAP) desensitizing agents. Materials and Methods: This study included 10 healthy fluorosed teeth and 10 nonfluorosed teeth with ages ranging from 20 to 40 years. Test Group 1 received 1% n-HAP desensitizing agent, whereas test Group 2 received 10% n-HAP formulation. Over 7 days, the dentin samples were brushed twice daily for 2 min. The samples were subsequently exposed to artificial saliva for 1 week. The diameter of the dentinal tubules was measured using ImageJ, and the dentinal tubules were manually counted using Adobe Photoshop CS3. Results and Discussion: When comparing the quantity and percentage of completely and partially occluded dentinal tubules in teeth treated with 1% and 10% n-HAP desensitizing agents, no statistically significant differences between fluorosed and nonfluorosed teeth were found (P = 0.94). Furthermore, the mean diameter of partially occluded dentinal tubules in fluorosed and nonfluorosed teeth was statistically significant (P = 0.00). Conclusion: When n-HAP toothpaste was used, dentinal tubules were occluded. Data from this trial and other studies indicate the therapeutic efficacy of the n-HAP dentifrice, suggesting that it could be utilized to reduce dentin hypersensitivity.

Teeth are considered to be the prime organs for mastication, speech, and esthetics. The basic structure of tooth involves enamel, dentin, and pulp. Each of these is formed by specialized structures during the various phases of tooth development. One of such specialized structures forming dentin of the tooth are odontoblasts. Being present at the periphery of the pulp chamber, they are in close contact with dentin through cytoplasmic extensions which travel into the entire length of dentin through dentinal tubules. In addition to dentin formation, they play a vital role in performing the various functions of the tooth including nerve sensation. They are functionally active throughout the life span of a healthy tooth and are subjective to numerous injuries causing various oral lesions. This review aims to collect the information with an additional note on the role of odontoblasts and their characteristics in a healthy and diseased tooth structure, their significance in endodontics and three-dimensional (3D) histological means.

Regenerative endodontic procedures (REPs) are promising for dental pulp tissue regeneration; however, their application in permanent teeth remains challenging. We assessed the potential combination of an REP and local dental pulp cell (DPC) transplantation in the mature molars of C57BL/6 mice with (REP + DPC group) or without (REP group) transplantation of DPCs from green fluorescent protein (GFP) transgenic mice. After 4 weeks, the regenerated tissue was evaluated by micro-computed tomography and histological analyses to detect odontoblasts, vasculogenesis, and neurogenesis. DPCs were assessed for mesenchymal and pluripotency markers. Four weeks after the REP, the molars showed no signs of periapical lesions, and both the REP and REP + DPC groups exhibited a pulp-like tissue composed of a cellular matrix with vessels surrounded by an eosin-stained acellular matrix that resembled hard tissue. However, the REP + DPC group had a broader cellular matrix and uniquely contained odontoblast-like cells co-expressing GFP. Vasculogenesis and neurogenesis were detected in both groups, with the former being more prominent in the REP + DPC group. Overall, the REP was achieved in mature mouse molars and DPC transplantation improved the outcomes by inducing the formation of odontoblast-like cells and greater vasculogenesis.

Mechanosensory ion channels are proteins that are sensitive to mechanical forces. They are found in tissues throughout the body and play an important role in bone remodeling by sensing changes in mechanical stress and transmitting signals to bone-forming cells. Orthodontic tooth movement (OTM) is a prime example of mechanically induced bone remodeling. However, the cell-specific role of the ion channels Piezo1 and Piezo2 in OTM has not been investigated yet. Here we first identify the expression of PIEZO1/2 in the dentoalveolar hard tissues. Results showed that PIEZO1 was expressed in odontoblasts, osteoblasts, and osteocytes, while PIEZO2 was localized in odontoblasts and cementoblasts. We therefore used a Piezo1floxed/floxed mouse model in combination with Dmp1cre to inactivate Piezo1 in mature osteoblasts/cementoblasts, osteocytes/cementocytes, and odontoblasts. Inactivation of Piezo1 in these cells did not affect the overall morphology of the skull but caused significant bone loss in the craniofacial skeleton. Histological analysis revealed a significantly increased number of osteoclasts in Piezo1floxed/floxed;Dmp1cre mice, while osteoblasts were not affected. Despite this increased number of osteoclasts, orthodontic tooth movement was not altered in these mice. Our results suggest that despite Piezo1 being crucial for osteoclast function, it may be dispensable for mechanical sensing of bone remodeling. [ABSTRACT FROM AUTHOR]

Odontoblasts are involved in sensory generation as sensory receptor cells and in dentin formation. We previously reported that an increase in intracellular cAMP levels by cannabinoid 1 receptor activation induces Ca2+ influx via transient receptor potential vanilloid subfamily member 1 channels in odontoblasts, indicating that intracellular cAMP/Ca2+ signal coupling is involved in dentinal pain generation and reactionary dentin formation. Here, intracellular cAMP dynamics in cultured human odontoblasts were investigated to understand the detailed expression patterns of the intracellular cAMP signaling pathway activated by the Gs protein-coupled receptor and to clarify its role in cellular functions. The presence of plasma membrane Gαs as well as prostaglandin I2 (IP), 5-hydroxytryptamine 5-HT4 (5-HT4), dopamine D1 (D1), adenosine A2A (A2A), and vasoactive intestinal polypeptide (VIP) receptor immunoreactivity was observed in human odontoblasts. In the presence of extracellular Ca2+, the application of agonists for the IP (beraprost), 5-HT4 (BIMU8), D1 (SKF83959), A2A (PSB0777), and VIP (VIP) receptors increased intracellular cAMP levels. This increase in cAMP levels was inhibited by the application of the adenylyl cyclase (AC) inhibitor SQ22536 and each receptor antagonist, dose-dependently. These results suggested that odontoblasts express Gs protein-coupled IP, 5-HT4, D1, A2A, and VIP receptors. In addition, activation of these receptors increased intracellular cAMP levels by activating AC in odontoblasts. [ABSTRACT FROM AUTHOR]

Dentin is the major hard tissue of teeth formed by differentiated odontoblasts. How odontoblast differentiation is regulated remains enigmatic. Here, we report that the E3 ubiquitin ligase CHIP is highly expressed in undifferentiated dental mesenchymal cells and downregulated after differentiation of odontoblasts. Ectopic expression of CHIP inhibits odontoblastic differentiation of mouse dental papilla cells, whereas knockdown of endogenous CHIP has opposite effects. Chip (Stub1) knockout mice display increased formation of dentin and enhanced expression of odontoblast differentiation markers. Mechanistically, CHIP interacts with and induces K63 polyubiquitylation of the transcription factor DLX3, leading to its proteasomal degradation. Knockdown of DLX3 reverses the enhanced odontoblastic differentiation caused by knockdown of CHIP. These results suggest that CHIP inhibits odontoblast differentiation by targeting its tooth-specific substrate DLX3. Furthermore, our results indicate that CHIP competes with another E3 ubiquitin ligase, MDM2, that promotes odontoblast differentiation by monoubiquitylating DLX3. Our findings suggest that the two E3 ubiquitin ligases CHIP and MDM2 reciprocally regulate DLX3 activity by catalyzing distinct types of ubiquitylation, and reveal an important mechanism by which differentiation of odontoblasts is delicately regulated by divergent post-translational modifications. [ABSTRACT FROM AUTHOR]

Dentin-pulp complex is object of interest in the regenerative endodontic field as well as the natural function of human dental pulp stem cells (hDPSCs) that may differentiate into specific cells able to repair and/or regenerate both hard and soft dental structures. The aim of the present study was to evaluate the capacity of hDPSCs to differentiate in odontoblastic-like cells by evaluating the expression of specific odontogenic-related genes and to prove the ability of treatment with calcium-based materials such as calcium carbonate (CaCO3), calcium hydroxide (Ca(OH)₂), and mineral trioxide aggregate (MTA). hDPSCs were obtained and isolated from a third molar of a young patient. Odontogenic-related gene expression was assessed unti1 28 days of culture as well as alkaline phosphatase activity (ALP). hDPSCs were cultured in odontoblastic-induction medium used as control, and in presence of different concentrations of CaCO 3, Ca(OH)₂, and MTA. The results demonstrated an upregulation in odontoblastic cell-related genes, in particular of the early differentiation marker known as matrix extracellular phosphoglycoprotein (MEPE), as well as increased ALP activity and the presence of calcium deposits, mainly by stimulation with calcium derivatives. In this regard, treatment of pulp tissue with CaCO 3 , Ca(OH) 2 and even better with MTA seemed to be effective for dentinogenesis. The ease of isolation of hDPSCs from discarded or extracted teeth offers a promising source of autologous cells that may be applied for regenerative purpose in combination with selected bioactive materials. However, further investigations should be conducted to confirm the obtained results. • CaCO 3 , Ca(OH) 2 and especially MTA seemed to be effective to promote dentinogenesis. • calcium-based materials induced differentiation of DPSC in odontoblastic-like cells. • During differentiation DPSC expressed specific odontogenic-related genes. • DPSC are promising for various regenerative medical applications. • Ca2+ had a significant impact on cellular stimulation in an odontogenic pattern. [ABSTRACT FROM AUTHOR]

Introduction: Although there are many desensitizing medications on the market with promising results, their therapeutic advantages are sometimes fleeting or diminished over time since they might be gradually destroyed by consistent tooth brushing or ingestion of acidic beverages. Aim: In this scanning electron microscope study, an initial effort was made to evaluate and compare the dentinal changes that occurred after healthy fluorosed and nonfluorosed teeth were subjected to the application of 1% and 10% nano-hydroxyapatite (n-HAP) desensitizing agents. Materials and Methods: This study included 10 healthy fluorosed teeth and 10 nonfluorosed teeth with ages ranging from 20 to 40 years. Test Group 1 received 1% n-HAP desensitizing agent, whereas test Group 2 received 10% n-HAP formulation. Over 7 days, the dentin samples were brushed twice daily for 2 min. The samples were subsequently exposed to artificial saliva for 1 week. The diameter of the dentinal tubules was measured using ImageJ, and the dentinal tubules were manually counted using Adobe Photoshop CS3. Results and Discussion: When comparing the quantity and percentage of completely and partially occluded dentinal tubules in teeth treated with 1% and 10% n-HAP desensitizing agents, no statistically significant differences between fluorosed and nonfluorosed teeth were found (P = 0.94). Furthermore, the mean diameter of partially occluded dentinal tubules in fluorosed and nonfluorosed teeth was statistically significant (P = 0.00). Conclusion: When n-HAP toothpaste was used, dentinal tubules were occluded. Data from this trial and other studies indicate the therapeutic efficacy of the n-HAP dentifrice, suggesting that it could be utilized to reduce dentin hypersensitivity. [ABSTRACT FROM AUTHOR]

Dentin sialophosphoprotein (DSPP) is primarily expressed by differentiated odontoblasts (dentin-forming cells), and transiently expressed by presecretory ameloblasts (enamel-forming cells). Disease-causing DSPP mutations predominantly fall into two categories: 5' mutations affecting targeting and trafficking, and 3' − 1 frameshift mutations converting the repetitive, hydrophilic, acidic C-terminal domain into a hydrophobic one. We characterized the dental phenotypes and investigated the pathological mechanisms of DsppP19L and Dspp−1fs mice that replicate the two categories of human DSPP mutations. In DsppP19L mice, dentin is less mineralized but contains dentinal tubules. Enamel mineral density is reduced. Intracellular accumulation and ER retention of DSPP is observed in odontoblasts and ameloblasts. In Dspp−1fs mice, a thin layer of reparative dentin lacking dentinal tubules is deposited. Odontoblasts show severe pathosis, including intracellular accumulation and ER retention of DSPP, strong ubiquitin and autophagy activity, ER-phagy, and sporadic apoptosis. Ultrastructurally, odontoblasts show extensive autophagic vacuoles, some of which contain fragmented ER. Enamel formation is comparable to wild type. These findings distinguish molecular mechanisms underlying the dental phenotypes of DsppP19L and Dspp−1fs mice and support the recently revised Shields classification of dentinogenesis imperfecta caused by DSPP mutations in humans. The Dspp−1fs mice may be valuable for the study of autophagy and ER-phagy. [ABSTRACT FROM AUTHOR]

Iroquois homeobox (Irx) genes are TALE-class homeobox genes that are evolutionarily conserved across species and have multiple critical cellular functions in fundamental tissue development processes. Previous studies have shown that Irxs genes are expressed during tooth development. However, the precise roles of genes in teeth remain unclear. Here, we demonstrated for the first time that Irx3 is an essential molecule for the proliferation and differentiation of odontoblasts. Using cDNA synthesized from postnatal day 1 (P1) tooth germs, we examined the expression of all Irx genes (Irx1 - Irx6) by RT-PCR and found that all genes except Irx4 were expressed in the tooth tissue. Irx1 - Irx3 a were expressed in the dental epithelial cell line M3H1 cells, while Irx3 and Irx5 were expressed in the dental mesenchymal cell line mDP cells. Only Irx3 was expressed in both undifferentiated cell lines. Immunostaining also revealed the presence of IRX3 in the dental epithelial cells and mesenchymal condensation. Inhibition of endogenous Irx3 by siRNA blocks the proliferation and differentiation of mDP cells. Wnt3a , Wnt5a , and Bmp4 are factors involved in odontoblast differentiation and were highly expressed in mDP cells by quantitative PCR analysis. Interestingly, the expression of Wnt5a (but not Wnt3a or Bmp4) was suppressed by Irx3 siRNA. These results suggest that Irx3 plays an essential role in part through the regulation of Wnt5a expression during odontoblast proliferation and differentiation. • Iroquois homeobox (Irx) genes are involved in tooth development. • Irx3 is an essential molecule for odontoblast proliferation and differentiation. • Irx3 regulates Wnt5a expression, a critical factor for odontoblast differentiation. [ABSTRACT FROM AUTHOR]

The current development of microfluidics-based microphysiological systems (MPSs) will rapidly lead to a paradigm shift from traditional static 2-dimensional cell cultivation towards organized tissue culture within a dynamic cellular milieu. Especially organs-on-a-chip (OoCs) can very precisely re-create the mechanical and unique anatomical structures of the oral environment. This review provides an introduction to such technology, from commonly used chip materials and fabrication methods to the application of OoC in in vitro culture. OoCs are advantageous because of their small-scaled culture environment, the highly controlled dynamic experimental conditions, and the likeness to the in vivo structure. We specifically focus on current chip designs in dental, oral, and craniofacial (DOC) research. Also, future perspectives are discussed, like model standardization and the development of integrated platforms with advanced read-out functionality. By doing so, it will be possible for OoCs to serve as an alternative for animal testing and to develop highly predictive human models for clinical experiments and even personalized medicine. [ABSTRACT FROM AUTHOR]

Understanding cell types and mechanisms of dental growth is essential for reconstruction and engineering of teeth. Therefore, we investigated cellular composition of growing and non-growing mouse and human teeth. As a result, we report an unappreciated cellular complexity of the continuously-growing mouse incisor, which suggests a coherent model of cell dynamics enabling unarrested growth. This model relies on spatially-restricted stem, progenitor and differentiated populations in the epithelial and mesenchymal compartments underlying the coordinated expansion of two major branches of pulpal cells and diverse epithelial subtypes. Further comparisons of human and mouse teeth yield both parallelisms and differences in tissue heterogeneity and highlight the specifics behind growing and non-growing modes. Despite being similar at a coarse level, mouse and human teeth reveal molecular differences and species-specific cell subtypes suggesting possible evolutionary divergence. Overall, here we provide an atlas of human and mouse teeth with a focus on growth and differentiation.

The gastropod radula is highly diverse in structure in comparison with that of other mollusсs. The main radular types in the different phylogenetic groups of gastropods differ not only in the general morphology and configuration of the teeth but also in the mode of tooth synthesis and the ultrastructure of the formation zone. Previously, the formation and anlage of the radula in the ontogeny of radulae of all major phylogenetic gastropods groups have been studied, with the exception of the taenioglossan radula of Caenogastropoda. The data obtained in this study on the radular anlage and synthesis in one littorinimorph species Lacuna pallidula supplement the existing knowledge of diversity in gastropod radula formation. The radula is initially formed at the stage of the post-torsion veliger, with five teeth in each transverse row, and acquires the adult morphology before hatching from the egg mass. The larval radula is synthesized by a few morphologically uniform cells in the radular sac. Synthesis of the adult radula also occurs at the blind end of the radular sac, where groups of numerous odontoblasts each form one tooth, and membranoblasts, located on the ventral side, form the membrane. Characteristic features of the adult radular sac are an additional supporting cartilage-like structure at the radula curve in the middle of the radular sac, a well-defined proliferation zone in the zone of radula formation and the presence of an additional extracellular matrix around the teeth in the maturation zone. [ABSTRACT FROM AUTHOR]

The (-)-Epigallocatechin-gallate (EGCG) metabolite is a natural polyphenol derived from green tea and is associated with antioxidant, biocompatible, and anti-inflammatory effects. Objective: To evaluate the effects of EGCG to promote the odontoblast-like cells differentiated from human dental pulp stem cells (hDPSCs); the antimicrobial effects on Escherichia coli, Streptococcus mutans, and Staphylococcus aureus; and improve the adhesion on enamel and dentin by shear bond strength (SBS) and the adhesive remnant index (ARI). Material and methods: hDSPCs were isolated from pulp tissue and immunologically characterized. EEGC dose-response viability was calculated by MTT assay. Odontoblast-like cells were differentiated from hDPSCs and tested for mineral deposition activity by alizarin red, Von Kossa, and collagen/vimentin staining. Antimicrobial assays were performed in the microdilution test. Demineralization of enamel and dentin in teeth was performed, and the adhesion was conducted by incorporating EGCG in an adhesive system and testing with SBS-ARI. The data were analyzed with normalized Shapiro–Wilks test and ANOVA post hoc Tukey test. Results: The hDPSCs were positive to CD105, CD90, and vimentin and negative to CD34. EGCG (3.12 µg/mL) accelerated the differentiation of odontoblast-like cells. Streptococcus mutans exhibited the highest susceptibility < Staphylococcus aureus < Escherichia coli. EGCG increased (p < 0.05) the dentin adhesion, and cohesive failure was the most frequent. Conclusion: (-)-Epigallocatechin-gallate is nontoxic, promotes differentiation into odontoblast-like cells, possesses an antibacterial effect, and increases dentin adhesion. [ABSTRACT FROM AUTHOR]

Objective: Evaluate the influence of a polymeric catalyst primer (PCP) on esthetic efficacy (EE), degradation kinetics of hydrogen peroxide (H2O2), and trans‐amelodentinal cytotoxicity (TC) of bleaching gels. Materials and Methods: The following groups were established: G1: No treatment (NC, negative control); G2: PCP; G3: 10% H2O2; G4: PCP + 10% H2O2; G5: 20% H2O2; G6: PCP + 20% H2O2; G7: 35% H2O2 (positive control); G8: PCP + 35% H2O2. To determine EE, enamel/dentin discs (E/DDs) were stained and subjected or not to bleaching protocols for 45 min. To assess TC, the E/DDs were adapted to artificial pulp chambers. The extracts (culture medium + gel components diffused through E/DDs) were applied to odontoblast‐like MDPC‐23 cells. The viability (VB), oxidative stress (OxS), morphology (SEM), amount of H2O2 diffused and the production of hydroxyl radical (OH•) were assessed (two‐way ANOVA/Tukey/paired Student t‐test; p < 0.05). Results: The highest EE was found in G8 (p < 0.05), and G4, G6, and G7 did not differ statistically (p > 0.05). In G4, the limited H2O2 diffusion reduced OxS and increased cell VB (p < 0.05). Conclusions: Coating the enamel with PCP containing 10 mg/ml of manganese oxide before applying the 10% H2O2 bleaching gel maintains the EE of conventional in‐office bleaching and minimizes the toxic effects of this esthetic therapy. Clinical Significance: Coating the enamel with a PCP before applying the bleaching gel may potentiate the EE of the conventional in‐office tooth bleaching and reduce the toxicity of this professional therapy to the dental pulp. [ABSTRACT FROM AUTHOR]

In human teeth with tooth decay and pulpitis, we found TRPC5 to be largely upregulated in sensory nerves, which will make TRPC5 a very efficient target for treating inflammatory tooth pain and dental hypersensitivity. In our experiments, TRPC5-deficient mice were the only cold transducer knockouts to lack signs of inflammatory tooth pain in a behavioral surrogate model of tooth pain based on sucrose consumption. In line with the pharmacological experiments, tooth nociceptors in jaw preparations derived from TRPC5/TRPA1 double null mice had only few cold nociceptors and all had much reduced response magnitudes and firing rates. [Extracted from the article]

This study aimed to evaluate the effects of flavonoids incorporated into poly(N-vinylcaprolactam) (PNVCL) hydrogel on cell viability and mineralization markers of odontoblast-like cells. MDPC-23 cells were exposed to ampelopsin (AMP), isoquercitrin (ISO), rutin (RUT) and control calcium hydroxide (CH) for evaluation of cell viability, total protein (TP) production, alkaline phosphatase (ALP) activity and mineralized nodule deposition by colorimetric assays. Based on an initial screening, AMP and CH were loaded into PNVCL hydrogels and had their cytotoxicity and effect on mineralization markers determined. Cell viability was above 70% when MDPC-23 cells were treated with AMP, ISO and RUT. AMP showed the highest ALP activity and mineralized nodule deposition. Extracts of PNVCL+AMP and PNVCL+CH in culture medium (at the dilutions of 1/16 and 1/32) did not affect cell viability and stimulated ALP activity and mineralized nodules' deposition, which were statistically higher than the control in osteogenic medium. In conclusion, AMP and AMP-loaded PNVCL hydrogels were cytocompatible and able to induce bio-mineralization markers in odontoblast-cells. [ABSTRACT FROM AUTHOR]

Teeth are considered to be the prime organs for mastication, speech, and esthetics. The basic structure of tooth involves enamel, dentin, and pulp. Each of these is formed by specialized structures during the various phases of tooth development. One of such specialized structures forming dentin of the tooth are odontoblasts. Being present at the periphery of the pulp chamber, they are in close contact with dentin through cytoplasmic extensions which travel into the entire length of dentin through dentinal tubules. In addition to dentin formation, they play a vital role in performing the various functions of the tooth including nerve sensation. They are functionally active throughout the life span of a healthy tooth and are subjective to numerous injuries causing various oral lesions. This review aims to collect the information with an additional note on the role of odontoblasts and their characteristics in a healthy and diseased tooth structure, their significance in endodontics and three-dimensional (3D) histological means. [ABSTRACT FROM AUTHOR]