Odontogenic Markers Research Articles

Comparative Immunohistochemical Analysis of Craniopharyngioma and Ameloblastoma: Insights into Odontogenic Differentiation.

Histopathological similarities between craniopharyngioma (CP) and ameloblastoma (AB) have long been recognized, particularly the shared features of palisading columnar epithelium and stellate reticulum-like areas. This study aimed to investigate potential odontogenic differentiation in CP akin to AB using immunohistochemical odontogenic markers. We analyzed AMELX, ODAM, and CK19 expression in 44 cases (20 CP and 24 AB). While AMELX and ODAM showed diffuse strong positive expression in both tumors with no significant statistical differences, CK19 expression was notably higher in CP. The markers AMELX and ODAM associated with odontogenic differentiation exhibited similar profiles in both tumors due to shared similar embryological origins. Notably, CK19, a biomarker of odontogenic epithelium, showed even higher expression, suggesting distinct pathways. These findings offer insights into tumor biology and may aid in diagnostic and therapeutic approaches.

Comprehensive analysis of transcription factors involved in odontoblast differentiation mechanism.

Primary cultured odontoblasts rapidly lose their tissue-specific phenotype. To identify transcription factors (TF) that are important for the maintenance of the odontoblast phenotype, primary cultures of C57BL/6J mouse dental mesenchymal cells (DMC) were isolated, and expression of TF and odontoblast marker genes in cells immediately after isolation and 2days after culture were comprehensively evaluated and compared using RNA-sequencing (RNA-seq). The expression of odontoblast markers in mouse dental mesenchymal cells decreased rapidly after isolation. In addition, the expression of Hedgehog-related, Notch-related, and immediate- early gene (IEG)-related transcription factors significantly decreased. Forced expression of these genes in lentiviral vectors, together with fibroblast growth factor 4 (FGF4), fibroblast growth factor 9 (FGF9), and the Wnt pathway activator CHIR99021, significantly induced the expression of odontogenic marker genes. These results indicate, for the first time, that Notch signaling and early genes may be important for maintaining odontoblast cultures. Furthermore, simultaneous stimulation of FGF, Wnt, Hedgehog, Notch pathways, and IEG transcription factors cooperatively promoted the maintenance of the odontoblast phenotype. These results suggest that the Hedgehog and Notch signaling pathways may play an important role in maintaining odontoblast phenotypes, in addition to FGF and Wnt signaling.

Biomimetic pulp scaffolds prepared from extracellular matrix derived from stem cells from human exfoliated deciduous teeth promote pulp-dentine complex regeneration.

To evaluate the role of biomimetic pulp scaffolds derived from the extracellular matrix derived of stem cells from human exfoliated deciduous teeth (SHED-ECM-PS) in promoting pulp-dentine complex regeneration. SHED-ECM-PS was prepared through cell aggregation and decellularization techniques. Histological and immunofluorescence analyses, scanning electron microscopy, and DNA quantification assays were used to characterize the SHED-ECM-PS. Additionally, a tooth slice implantation model was established to evaluate the effects of SHED-ECM-PS on regeneration of the pulp-dentine complex invivo. Extraction medium for SHED-ECM-PS was prepared, and its effect on bone marrow mesenchymal stem cells (BMMSCs) was assessed invitro. Cell counting kit-8 and Ki-67 staining assays were performed to determine cell proliferation. The rate of apoptosis was evaluated by flow cytometry. Wound healing and transwell assays were conducted to evaluate cell migration. Alizarin red S staining was performed to examine mineralized nodule formation. Western blotting was used to detect the expression of osteogenic and odontogenic markers. The results were analysed using an independent two-tailed Student's t-test. p < .05 was considered statistically significant. SHED-ECM-PS was successfully constructed, exhibiting a striped dental pulp-like shape devoid of nuclear structures or DNA components, and rich in fibronectin, collagen I, DMP1 and DSPP. Notably, SHED-ECM-PS showed no impact on the proliferation or apoptosis of BMMSCs. Histological analysis revealed that dental pulp fibroblasts formed an interwoven mesh in the root canal, and angiogenesis was observed in the SHED-ECM-PS group. Moreover, a continuous, newly formed tubular dentine layer with polarized odontoblast-like cells was observed along the inner wall of the root canal. SHED-ECM-PS promoted the migration, polar alignment and mineralized nodule formation of BMMSCs and specifically elevated the expression levels of odontogenic markers, but not osteogenic markers, compared with the control group invitro. SHED-ECM-PS exhibited no cytotoxicity and promoted pulp-dentine complex regeneration invivo as well as cell migration and odontogenic differentiation of BMMSCs invitro. These findings provide evidence that SHED-ECM-PS, as a novel biological scaffold, has the potential to improve the outcomes of REPs.

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

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

Functionalization of PCL-Based Fiber Scaffolds with Different Sources of Calcium and Phosphate and Odontogenic Potential on Human Dental Pulp Cells.

This study investigated the incorporation of sources of calcium, phosphate, or both into electrospun scaffolds and evaluated their bioactivity on human dental pulp cells (HDPCs). Additionally, scaffolds incorporated with calcium hydroxide (CH) were characterized for degradation, calcium release, and odontogenic differentiation by HDPCs. Polycaprolactone (PCL) was electrospun with or without 0.5% w/v of calcium hydroxide (PCL + CH), nano-hydroxyapatite (PCL + nHA), or β-glycerophosphate (PCL + βGP). SEM/EDS analysis confirmed fibrillar morphology and particle incorporation. HDPCs were cultured on the scaffolds to assess cell viability, adhesion, spreading, and mineralized matrix formation. PCL + CH was also evaluated for gene expression of odontogenic markers (RT-qPCR). Data were submitted to ANOVA and Student's t-test (α = 5%). Added CH increased fiber diameter and interfibrillar spacing, whereas βGP decreased both. PCL + CH and PCL + nHA improved HDPC viability, adhesion, and proliferation. Mineralization was increased eightfold with PCL + CH. Scaffolds containing CH gradually degraded over six months, with calcium release within the first 140 days. CH incorporation upregulated DSPP and DMP1 expression after 7 and 14 days. In conclusion, CH- and nHA-laden PCL fiber scaffolds were cytocompatible and promoted HDPC adhesion, proliferation, and mineralized matrix deposition. PCL + CH scaffolds exhibit a slow degradation profile, providing sustained calcium release and stimulating HDPCs to upregulate odontogenesis marker genes.

Photobiomodulation and low-intensity pulsed ultrasound synergistically enhance dental mesenchymal stem cells viability, migration and differentiation: an invitro study

Novel methods and technologies that improve mesenchymal stem cells (MSCs) proliferation and differentiation properties are required to increase their clinical efficacy. Photobiomodulation (PBM) and low-intensity pulsed ultrasound (LIPUS) are two strategies that can be used to enhance the regenerative properties of dental MSCs. This study evaluated the cytocompatibility and osteo/odontogenic differentiation of dental pulp, periodontal ligament, and gingival MSCs after stimulation by either PBM or LIPUS and their combined effect. MTT assay, cell migration assay, osteo/odontogenic differentiation by AR staining and ALP activity, and expression of osteo/odontogenic markers (OPG, OC, RUNX2, DSPP, DMP1) by RT-qPCR were evaluated. Statistical analysis was performed using ANOVA, followed by Tukey’s post hoc test, with a p-value of less than 0.05 considered significant. The results showed that combined stimulation by PBM and LIPUS resulted in significantly the highest viability of MSCs, the fastest migration, the most dense AR staining, the most increased ALP activity, and the most elevated levels of osteogenic and odontogenic markers. The synergetic stimulation of PBM and LIPUS can be utilized in cell-based regenerative approaches to promote the properties of dental MSCs.

Effect of polydatin on the viability and odontogenic differentiation of human dental pulp stem cells: An in-vitro study

Background/purposeVarious materials have been used to promote human dental pulp stem cells (hDPSCs) differentiation to produce dentin bridge formation with less-than-optimal results. Polydatin (PD), a naturally present material with osteogenic properties can be a promising material in the pulp regeneration/repair process. The aim of this study was to evaluate the effect of (PD) on the viability and differentiation of human dental pulp stem cells. Materials and methodsPD effect on hDPSCs in terms of cellular viability, alkaline phosphatase (ALP) production, and messenger RNAs (mRNA) of odontogenic markers production using quantitative reverse transcription polymerase chain reaction (RT-qPCR) were evaluated. In addition, mineral deposits were detected with Alizarin red stain. ResultsThe viable hDPSCs in the presence of 0.01 μM and 0.1 μM PD were significantly higher than the control on days 3 and 7, respectively. In addition, ALP activity of hDPSCs was significantly increased with 0.01, 0.1, and 1 μM of PD. In addition, increased expression mRNAs of ALP, osteocalcin (OC), osteonectin (ON), osteopontin (OP), Runt-related transcription factor-2 (RUNX-2), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) was observed after PD treatment, however, the difference was not statistically significant. Furthermore, increased size of mineral deposits was observed with PD. ConclusionPD promoted the expression of markers associated with odontogenic differentiation and mineralized tissue deposition in hDPSCs.

Evaluation of the genotoxicity, cytotoxicity, and bioactivity of calcium silicate-based cements

BackgroundAs calcium silicate-based cements (CSCs) have found success in various vital pulp therapy applications, several new CSC products have emerged. This study aimed to assess the genotoxicity, cytotoxicity, and bioactivity of four CSCs by comparing the newly introduced materials Bio MTA+ and MTA Cem with previously studied materials, Biodentine and NeoMTA.MethodsGenotoxicity was evaluated using the micronucleus (MN) assay in human peripheral blood lymphocyte cells, measuring MN frequency and nuclear division index (NDI). Cytotoxicity was assessed in human dental pulp stem cells through the Water-Soluble Tetrazolium Salt-1 (WST-1) colorimetric assay. Bioactivity was determined by ELISA, measuring the levels of angiogenic and odontogenic markers (BMP-2, FGF-2, VEGF, and ALP). Statistical analyses included ANOVA, Dunnet and Sidak tests, and Wald chi-square test. (p < .05).ResultsThe MN frequency in the groups was significantly lower than that in the positive control group (tetraconazole) (p < .05). NDI values decreased with increasing concentration (p < .05). Bio MTA+ and NeoMTA showed decreased cell viability at all concentrations in 7-day cultures (p < .01). All materials increased BMP-2, FGF-2, and VEGF levels, with Biodentine and NeoMTA showing the highest levels of BMP-2 and FGF-2 on day 7. Biodentine displayed the highest VEGF levels on day 7. Biodentine and NeoMTA groups exhibited significantly higher ALP activity than the Bio MTA+ and MTA Cem groups by day 7.ConclusionBio MTA+ and MTA Cem demonstrated no genotoxic or cytotoxic effects. Moreover, this study revealed bioactive potentials of Bio MTA+ and MTA Cem by enhancing the expression of angiogenic and osteogenic growth factors.

Bone or Tooth dentin: The TGF-β signaling is the key.

To investigate the cell linkage between tooth dentin and bones, we studied TGF-β roles during postnatal dentin development using TGF-β receptor 2 (Tgfβr2) cKO models and cell lineage tracing approaches. Micro-CT showed that the early Tgfβr2 cKO exhibit short roots and thin root dentin (n = 4; p<0.01), a switch from multilayer pre-odontoblasts/odontoblasts to a single-layer of bone-like cells with a significant loss of ~85% of dentinal tubules (n = 4; p<0.01), and a matrix shift from dentin to bone. Mechanistic studies revealed a statistically significant decrease in odontogenic markers, and a sharp increase in bone markers. The late Tgfβr2 cKO teeth displayed losses of odontoblast polarity, a significant reduction in crown dentin volume, and the onset of massive bone-like structures in the crown pulp with high expression levels of bone markers and low levels of dentin markers. We thus concluded that bones and tooth dentin are in the same evolutionary linkage in which TGF-β signaling defines the odontogenic fate of dental mesenchymal cells and odontoblasts. This finding also raises the possibility of switching the pulp odontogenic to the osteogenic feature of pulp cells via a local manipulation of gene programs in future treatment of tooth fractures.

Effect of pulp capping materials on odontogenic differentiation of human dental pulp stem cells: An in vitro study.

Migration and differentiation of human dental pulp stem cells (hDPSCs) is a vital and key factor in the success of reparative dentin formation for maintenance of pulp vitality. Pulp capping materials are used to stimulate DPSCs to induce new dentin formation. Thus, the aim of the present study was to compare the response of DPSCs to four commercially available pulp capping materials: a bioactive bioceramic (Material 1), a nonresinous ready-to-use bioceramic cement (Material 2), a bioactive composite (Material 3),and a biocompatible, dual-cured, resin-modified calcium silicate (Material 4). hDPSCs were isolated and cultured from freshly extracted teeth and were then characterized by flow cytometry and multilineage differentiation. Discs prepared from pulp capping materials were tested with hDPSCs and MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, cell migration assay and odontogenic differentiation assay was performed. Expression of osteogenic markers (osteopontin, RUNX family transcription factor 2, osteocalcin) and the odontogenic marker (dentin sialophosphoprotein) was detected using reverse transcription-polymerase chain reaction. Materials 1, 2, and 3 generated more cell viability than Material 4. Furthermore, Material 4 showed the least wound exposure percentage, while Material 3 showed the highest percentage. Enhanced mineralization was found in hDSCPs cultured with Material 3,followed by Material 1, and then Material 2, while Material 4 revealed the least calcified mineralization. The results of this study were inconclusive regards contemporary bioceramic materials designed for vital pulp therapy as they have different effects on hDPSC. Further testing for cytotoxicity using live-dead staining, animal experiments, clinical trials, and independent analyses of these biomaterials is necessary for clinicians to make an informed decision for their use.

Novel Universal Bond Containing Bioactive Monomer Promotes Odontoblast Differentiation In Vitro.

The development of multifunctional materials has been expected in dentistry. This study investigated the effects of a novel universal bond containing a bioactive monomer, calcium 4-methacryloxyethyl trimellitic acid (CMET), on odontoblast differentiation in vitro. Eluates from bioactive universal bond with CMET (BA (+), BA bond), bioactive universal bond without CMET (BA (-)), and Scotchbond Universal Plus adhesive (SC, 3M ESPE, USA) were added to the culture medium of the rat odontoblast-like cell line MDPC-23. Then, cell proliferation, differentiation, and mineralization were examined. Statistical analyses were performed using a one-way ANOVA and Tukey's HSDtest. The cell counting kit-8 assay and alkaline phosphatase (ALP) assay showed that cell proliferation and ALP were significantly higher in the 0.5% BA (+) group than in the other groups. In a real-time reverse-transcription polymerase chain reaction, mRNA expression of the odontogenic markers, dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1), was significantly higher in the 0.5% BA (+) group than in the BA (-) and SC groups. Calcific nodule formation in MDPC-23 cells was accelerated in the BA (+) group in a dose-dependent manner (p < 0.01); however, no such effect was observed in the BA (-) and SC groups. Thus, the BA bond shows excellent potential for dentin regeneration.

Influence of Extracellular Matrix Components on the Differentiation of Periodontal Ligament Stem Cells in Collagen I Hydrogel.

Regeneration of periodontal tissues requires an integrated approach to the restoration of the periodontal ligament, cementum, and alveolar bone surrounding the teeth. Current strategies in endogenous regenerative dentistry widely use biomaterials, in particular the decellularized extracellular matrix (dECM), to facilitate the recruitment of populations of resident cells into damaged tissues and stimulate their proliferation and differentiation. The purpose of our study was to evaluate the effect of the exogenous components of the extracellular matrix (hyaluronic acid, laminin, fibronectin) on the differentiation of periodontal ligament stem cells (PDLSCs) cultured with dECM (combinations of decellularized tooth matrices and periodontal ligament) in a 3D collagen I hydrogel. The immunohistochemical expression of various markers in PDLSCs was assessed quantitatively and semi-quantitatively on paraffin sections. The results showed that PDLSCs cultured under these conditions for 14 days exhibited phenotypic characteristics consistent with osteoblast-like and odontoblast-like cells. This potential has been demonstrated by the expression of osteogenic differentiation markers (OC, OPN, ALP) and odontogenic markers (DSPP). This phenomenon corresponds to the in vivo state of the periodontal ligament, in which cells at the interface between bone and cementum tend to differentiate into osteoblasts or cementoblasts. The addition of fibronectin to the dECM most effectively induces the differentiation of PDLSCs into osteoblast-like and odontoblast-like cells under 3D culture conditions. Therefore, this bioengineered construct has a high potential for future use in periodontal tissue regeneration.

Exploring the Effects of Low-Level Laser Therapy on the Cytocompatibility and Osteo/Odontogenic Potential of Gingival-Derived Mesenchymal Stem Cells: Preliminary Report

Numerous tissue engineering uses for gingival-derived mesenchymal stem cells (GMSCs) have been demonstrated. Recently, low-level laser therapy (LLLT) has been projected as a factor that can improve MSCs’ regeneration capacity. Therefore, the aim of this research was to examine the impact of LLLT at 1.5 J/cm2 and 3 J/cm2 on the viability and osteo/odontogenic potential of GMSCs. An MTT assay was performed to detect viability. Osteo/odontogenic differentiation was evaluated using Alizarin Red S staining and qRT-PCR for the evaluation of the RUNX2, OC, DMP1, and DSPP genes. A two-way ANOVA with Tukey’s post hoc test was used to determine the statistical significance between groups. The results revealed that LLLT of both energy densities had no cytotoxic effect on GMSC viability. LLLT of 1.5 J/cm2 demonstrated better viability than the higher energy density (3 J/cm2). Furthermore, the osteo/odontogenic differentiation potential was promoted following LLLT radiation, where both groups exhibited mineralized nodule formation, with the low-energy laser having a significantly higher Alizarin Red S stain level. A qRT-PCR analysis revealed higher expression levels of osteogenic and odontogenic markers in the LLLT groups compared to the control group. In conclusion, this study showed the potential application of LLLT as a non-toxic and effective strategy to enhance the regenerative capacity of GMSCs for tissue engineering and clinical treatments in the oral and craniofacial fields.

Kappa-Carrageenan/Chitosan/Gelatin Scaffolds Provide a Biomimetic Microenvironment for Dentin-Pulp Regeneration

This study aims to investigate the impact of kappa-carrageenan on dental pulp stem cells (DPSCs) behavior in terms of biocompatibility and odontogenic differentiation potential when it is utilized as a component for the production of 3D sponge-like scaffolds. For this purpose, we prepared three types of scaffolds by freeze-drying (i) kappa-carrageenan/chitosan/gelatin enriched with KCl (KCG-KCl) as a physical crosslinker for the sulfate groups of kappa-carrageenan, (ii) kappa-carrageenan/chitosan/gelatin (KCG) and (iii) chitosan/gelatin (CG) scaffolds as a control. The mechanical analysis illustrated a significantly higher elastic modulus of the cell-laden scaffolds compared to the cell-free ones after 14 and 28 days with values ranging from 25 to 40 kPa, showing an increase of 27-36%, with the KCG-KCl scaffolds indicating the highest and CG the lowest values. Cell viability data showed a significant increase from days 3 to 7 and up to day 14 for all scaffold compositions. Significantly increasing alkaline phosphatase (ALP) activity has been observed over time in all three scaffold compositions, while the KCG-KCl scaffolds indicated significantly higher calcium production after 21 and 28 days compared to the CG control. The gene expression analysis of the odontogenic markers DSPP, ALP and RunX2 revealed a two-fold higher upregulation of DSPP in KCG-KCl scaffolds at day 14 compared to the other two compositions. A significant increase of the RunX2 expression between days 7 and 14 was observed for all scaffolds, with a significantly higher increase of at least twelve-fold for the kappa-carrageenan containing scaffolds, which exhibited an earlier ALP gene expression compared to the CG. Our results demonstrate that the integration of kappa-carrageenan in scaffolds significantly enhanced the odontogenic potential of DPSCs and supports dentin-pulp regeneration.

Topographic Cues of a PLGA Scaffold Promote Odontogenic Differentiation of Dental Pulp Stem Cells through the YAP/β-Catenin Signaling Axis.

The underlying mechanism of how topographic cues of artificial scaffolds regulate cell function remains poorly understood. Yes-associated protein (YAP) and β-catenin signaling have both been reported to play important roles in mechano-transduction and dental pulp stem cells (DPSCs) differentiation. We investigated the effects of YAP and β-catenin in spontaneous odontogenic differentiation of DPSCs induced by topographic cues of a poly(lactic-co-glycolic acid) (PLGA) membrane. The topographic cues and function of a fabricated PLGA scaffold were explored via scanning electron microscopy (SEM), alizarin red staining (ARS), reverse transcription-polymerase chain reaction (RT-PCR), and pulp capping. Immunohistochemistry (IF), RT-PCR, and western blotting (WB) were used to observe the activation of YAP and β-catenin when DPSCs were cultured on the scaffolds. Further, YAP was inhibited or overexpressed on either side of the PLGA membrane, and YAP, β-catenin, and odontogenic marker expression were analyzed using IF, ARS, and WB. The closed side of the PLGA scaffold promoted spontaneous odontogenic differentiation and nuclear translocation of YAP and β-catenin in vitro and in vivo compared to the open side. The YAP antagonist verteporfin inhibited β-catenin expression, nuclear translocation, and odontogenic differentiation on the closed side, but the effects were rescued by LiCl. YAP overexpressing DPSCs on the open side activated β-catenin signaling and promoted odontogenic differentiation. The topographic cue of our PLGA scaffold promotes odontogenic differentiation of DPSCs and pulp tissue through the YAP/β-catenin signaling axis.

Effects of CTHRC1 on odontogenic differentiation and angiogenesis in human dental pulp stem cells.

This study aimed to determine whether collagen triple helix repeat containing-1 (CTHRC1), which is involved in vascular remodeling and bone formation, can stimulate odontogenic differentiation and angiogenesis when administered to human dental pulp stem cells (hDPSCs). The viability of hDPSCs upon exposure to CTHRC1 was assessed with the WST-1 assay. CTHRC1 doses of 5, 10, and 20 µg/mL were administered to hDPSCs. Reverse-transcription polymerase reaction was used to detect dentin sialophosphoprotein, dentin matrix protein 1, vascular endothelial growth factor, and fibroblast growth factor 2. The formation of mineralization nodules was evaluated using Alizarin red. A scratch wound assay was conducted to evaluate the effect of CTHRC1 on cell migration. Data were analyzed using 1-way analysis of variance followed by the Tukey post hoc test. The threshold for statistical significance was set at p < 0.05. CTHRC1 doses of 5, 10, and 20 µg/mL had no significant effect on the viability of hDPSCs. Mineralized nodules were formed and odontogenic markers were upregulated, indicating that CTHRC1 promoted odontogenic differentiation. Scratch wound assays demonstrated that CTHRC1 significantly enhanced the migration of hDPSCs. CTHRC1 promoted odontogenic differentiation and mineralization in hDPSCs.

Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions.

Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.

Functional expression of oxytocin receptors in pulp-dentin complex

Dental pulp-derived stromal cells (DPSCs) are a crucial cell population for maintaining the tissue integrity of the pulp-dentin complex. The oxytocin receptor (OXTR), a member of the G protein-coupled receptor (GPCR) superfamily, plays versatile roles in diverse biological contexts. However, the role of OXTR in dental pulp has not yet been fully understood. Here, we demonstrate the biological functions and significance of OXTR in DPSCs through a multidisciplinary approach. Microarray data of 494 GPCR genes revealed high OXTR expression in human DPSCs (hDPSCs). Blocking OXTR activity increased the expression of osteogenic and odontogenic marker genes, promoting hDPSC differentiation. Additionally, we found that OXTR is involved in extracellular matrix (ECM) remodeling through the regulation of the gene expression related to ECM homeostasis. We further demonstrated that these genetic changes are mediated by trascriptional activity of Yes-associated protein (YAP). Based on the results, a preclinical experiment was performed using an animal model, demonstrating that the application of an OXTR inhibitor to damaged pulp induced significant hard tissue formation. These results provide new insight into the oxytocin-OXTR system in the regenerative process of pulp-dentin complex.

Dentin Particulate for Bone Regeneration: An In Vitro Study.

The aim of this in vitro study was to investigate the commitment and behavior of dental pulp stem cells (DPSCs) seeded onto two different grafting materials, human dentin particulate (DP) and deproteinized bovine bone matrix (BG), with those cultured in the absence of supplements. Gene expression analyses along with epigenetic and morphological tests were carried out to examine odontogenic and osteogenic differentiation and cell proliferation. Compressive testing of the grafting materials seeded with DPSCs was performed as well. DPSC differentiation into odontoblast-like cells was identified from the upregulation of odontogenic markers (DSPP and MSX) and osteogenic markers (RUNX2, alkaline phosphatase, osteonectin, osteocalcin, collagen type I, bmp2, smad5/8). Epigenetic tests confirmed the presence of miRNAs involved in odontogenic or osteogenic commitment of DPSCs cultured for up to 21 days on DP. Compressive strength values obtained from extracellular matrix (ECM) synthesized by DPSCs showed a trend of being higher when seeded onto DP than onto BG. High expression of VEGF factor, which is related to angiogenesis, and of dentin sialoprotein was observed only in the presence of DP. Morphological analyses confirmed the typical phenotype of adult odontoblasts. In conclusion, the odontogenic and osteogenic commitment of DPSCs and their respective functions can be achieved on DP, which enables exceptional dentin and bone regeneration.

Lead inhibits the odontogenic differentiation potential of dental pulp stem cells by affecting WNT1/β-catenin signaling and related miRNAs expression

Lead (Pb) is ubiquitous in environment that accumulates in teeth and calcified tissues from where it releases gradually with aging and adversely affects dental health. This study aimed to determine the effect of Pb exposure on odontogenic differentiation potential of isolated human dental pulp stem cells and investigate the possible underlying epigenetic factors. In the absence of Pb exposure, stem cells displayed significant odontogenic markers including elevated Alkaline Phosphatase (ALP) activity, Alizarin red staining intensity, and increased expression of odontogenic DMP1 and DSPP genes. Exposure to 60 μM Pb resulted in reduced ALP activity and calcium deposition. Also, diminished expression of RUNX2, DMP1, and DSPP, as well as Wnt signaling mediators including WNT1, and β-catenin were detected. The expression of Wnt signaling related microRNAs, miRNA-139-5p and miRNA-142-3p, on the other hand, were shown to have a significant increase. We concluded that Pb could adversely affect the odontogenic differentiation potential of dental pulp stem cell. The underlying mechanism might related to Pb-induced epigenetic dysregulation of WNT1/β-catenin pathway-related miRNAs leading to down-regulation of Wnt/β-catenin related odontogenic genes and eventually impaired odontogenic differentiation process.