AIM:The dental pulp is an immunologically active tissue that responds dynamically to cariogenic challenge. Peripheral pulp cells adjacent to dentine encounter bacterial stimuli earlier than cells located in the central pulp. To investigate signalling and immune interactions, this study profiled the transcriptomes of dentine-adherent cells (DACs) and central dental pulp cells (DPCs) cocultured with Streptococcus mutans. METHODOLOGY:Primary cultures of both DACs and DPCs were obtained from healthy third molars of three female and three male donors aged 13-16. Cells were cocultured with viable S. mutans (2 × 108 CFU/mL) for 6 h (n = 6). Controls included γ-inactivated bacteria and unexposed cells. RNA libraries (Illumina Stranded mRNA Prep) were sequenced on a NextSeq2000. Bioinformatic analysis included differential gene expression (DESeq2), gene set enrichment analysis (GSEA), and protein-protein interaction (PPI) network construction. Batch effects were corrected, and significantly regulated genes (|log2FC| > 1.5, padj < 0.05) were identified. Validation of DEGs was performed via reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS:RNA-Seq revealed a dynamic shift in the transcriptome of DACs and DPCs stimulated with S. mutans, while cells exposed to γ-inactivated or no bacteria did not. Although DACs and DPCs shared common DEGs (33 up, 8 down), several regulations were exclusive to DACs (22 up, 9 down) and DPCs (9 up, 25 down), highlighting a donor-independent functional specificity of the pulp subpopulations. Functional enrichment analysis revealed a strong and comparable activation of hypoxia-related pathways in both DPCs and DACs. However, DACs additionally showed enrichment in extracellular matrix organisation and cytokine signalling, while DPCs were characterised by intracellular stress responses and protein folding pathways. Additionally, protein-protein interaction analysis identified IL-6 as a key hub in DACs, while ANGPTL4 was central in DPCs. CONCLUSION:Following exposure to S. mutans, mechanically isolated DACs and DPCs displayed distinct transcriptomic profiles, indicating functional heterogeneity in the pulpal immune response. DACs engaged immunomodulatory pathways, while DPCs were marked by cellular stress responses, suggesting divergent contributions to tissue defence and homeostasis.

OBJECTIVE:This study aimed to investigate the inhibition of Toll-like receptor 2 (TLR2) signaling in lipoteichoic acid-induced NF-κB activation and interleukin-6 (IL-6) production in primary human dental pulp stromal cells (DPSCs). DESIGN:Primary human DPSCs were cultured with lipoteichoic acid from Staphylococcus aureus (LTA-SA; 10, 25, 50 µg/ml) for up to 72 h to assess time- and concentration-dependent IL-6 production. IL-6 secretion was quantified by ELISA, while cell counts were determined via cell counter. To evaluate TLR2-dependent signaling, cells were pre-incubated with the TLR2 inhibitor C29 (100 µM) prior to LTA-SA stimulation. IL6 gene expression was analyzed by RT-qPCR, and NF-κB nuclear translocation was assessed by Western blot analysis. Non-parametric statistical analyses were applied to compare all groups and time points (Mann-Whitney U test or Kruskal-Wallis test; α = 0.05). RESULTS:LTA-SA stimulation induced a significant, time- and concentration-dependent increase in IL6 gene expression and IL-6 secretion, accompanied by enhanced NF-κB nuclear translocation. Inhibition of TLR2 with C29 reduced nuclear translocation of NF-κB, along with a decrease in IL6 gene expression and IL-6 secretion, exhibiting both time- and concentration-dependent effects. CONCLUSIONS:LTA induces IL-6 production in DPSCs via TLR2-mediated activation of the canonical NF-κB pathway. Targeted modulation of TLR2 signaling may represent a potential strategy for controlling pulpal inflammation.

The incorporation of cerium instead of calcium into the crystal lattice of hydroxyapatite appears to increase the resistance of dental hard tissues to caries lesion initiation and progression. The effect on initial biofilm formation is yet unknown. The aim of this study was to assess the effect of cerium(III)chloride (50%CeCl3) pretreatment of hydroxyapatite (HA) discs on subsequent growth of an initial 3 species caries-biofilm. Twelve 9.5 mm diameter hydroxyapatite discs were divided into three groups (n = 4) and treated for 1 min with either 50% CeCl3, ultrapure water (Control), or 0.02% chlorhexidine gluconate (CHX) and washed twice in ultrapure water for 1 min. Samples were incubated in artificial saliva (21 °C, 120 min) for pellicle formation and then placed in an active attachment caries biofilm model comprising Actinomyces naeslundii, Schaalia odontolytica, and Streptococcus mutans, cultured anaerobically at 37 °C for 4 h before being fixed in 2.5% glutaraldehyde and examined using scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDX) in high-vacuum mode. SEM-micrographs at up to 50,000× showed net-like or spherical precipitates on the surface of all CeCl3-samples but not on the Control or CHX-samples. CeCl3-samples also showed signs of acid attack possibly due to the low pH (2.6) of the CeCl3 solution. Rods and cocci were found on all Control, but only on 2 of 4 CHX samples. On CeCl3 samples, only one harbored isolated cocci but no rods were observed. EDX-analyses confirmed the presence of Cerium in all CeCl3 samples with atomic percent (At%) ranging from 0.1 to 0.4 for areas without visible precipitates and up to 4.1 for areas with precipitates. CeCl3-treatment before pellicle formation results in the development of precipitates on the surface of HA and appears to have potential to inhibit initial biofilm growth on HA compared to CHX treated or untreated controls.

Fluoride-based cariostatic agents are commonly used in dental products and are generally considered safe. However, ongoing discussions about potential adverse effects are driving research into alternative agents, such as lanthanide salts. This study aims to evaluate the cytotoxic effects of different cariostatic agents, including fluoride compounds (NaF, Na2PO3F, NH4F) and lanthanide salts (Ce(NO3)3, CeCl3, Sm(NO3)3, SmCl3). Mouse fibroblasts (L-929) were cultured in Eagle’s Minimum Essential Medium supplemented with 5

AIM:To develop a new coculture system that allows exposure of dental pulp cells (DPCs) to Streptococcus mutans and dentine matrix proteins (eDMP) to study cellular interactions in dentine caries.METHODOLOGY:Dental pulp cells and S. mutans were cocultured with or without eDMP for 72 h. Cell proliferation and viability were assessed by cell counting and MTT assays, while bacterial growth and viability were determined by CFU and LIVE/DEAD staining. Glucose catabolism and lactate excretion were measured photometrically as metabolic indicators. To evaluate the inflammatory response, the release of cytokines and growth factors (IL-6, IL-8, TGF-β1, VEGF) was determined by ELISA. Non-parametric statistical analyses were performed to compare all groups and time points (Mann-Whitney U test or Kruskal-Wallis test; α = .05).RESULTS:While eDMP and especially S. mutans reduced the number and viability of DPCs (p ≤ .0462), neither DPCs nor eDMP affected the growth and viability of S. mutans during coculture (p > .0546). The growth of S. mutans followed a common curve, but the death phase was not reached within 72 h. S. mutans consumed medium glucose in only 30 h, whereas in the absence of S. mutans, cells were able to catabolize glucose throughout 72 h, resulting in the corresponding amount of l-lactate. No change in medium pH was observed. S. mutans induced IL-6 production in DPCs (p ≤ .0011), whereas eDMP had no discernible effect (p > .7509). No significant changes in IL-8 were observed (p > .198). TGF-β1, available from eDMP supplementation, was reduced by DPCs over time. VEGF, on the other hand, was increased in all groups during coculture.CONCLUSIONS:The results show that the coculture of DPCs and S. mutans is possible without functional impairment. The bacterially induced stimulation of proinflammatory and regenerative cytokines provides a basis for future investigations and the elucidation of molecular biological relationships in pulp defence against caries.

To develop a new coculture system that allows exposure of dental pulp cells (DPCs) to Streptococcus mutans and dentine matrix proteins (eDMP) to study cellular interactions in dentine caries. Dental pulp cells and S. mutans were cocultured with or without eDMP for 72 h. Cell proliferation and viability were assessed by cell counting and MTT assays, while bacterial growth and viability were determined by CFU and LIVE/DEAD staining. Glucose catabolism and lactate excretion were measured photometrically as metabolic indicators. To evaluate the inflammatory response, the release of cytokines and growth factors (IL-6, IL-8, TGF-β1, VEGF) was determined by ELISA. Non-parametric statistical analyses were performed to compare all groups and time points (Mann–Whitney U test or Kruskal–Wallis test; α  = .05). While eDMP and especially S. mutans reduced the number and viability of DPCs ( p  ≤ .0462), neither DPCs nor eDMP affected the growth and viability of S. mutans during coculture ( p  > .0546). The growth of S. mutans followed a common curve, but the death phase was not reached within 72 h. S. mutans consumed medium glucose in only 30 h, whereas in the absence of S. mutans , cells were able to catabolize glucose throughout 72 h, resulting in the corresponding amount of l -lactate. No change in medium pH was observed. S. mutans induced IL-6 production in DPCs ( p  ≤ .0011), whereas eDMP had no discernible effect ( p  > .7509). No significant changes in IL-8 were observed ( p  > .198). TGF-β1, available from eDMP supplementation, was reduced by DPCs over time. VEGF, on the other hand, was increased in all groups during coculture. The results show that the coculture of DPCs and S. mutans is possible without functional impairment. The bacterially induced stimulation of proinflammatory and regenerative cytokines provides a basis for future investigations and the elucidation of molecular biological relationships in pulp defence against caries.

The objective of this study was to compare the ability of different endodontic irrigation activation methods to enable irrigant penetration, remove the smear layer from root canal walls after preparation, and investigate surface effects on dentine. Root canals of 90 single-rooted teeth were prepared and irrigated with EDTA (17%) and sodium hypochlorite (5%), where both irrigants or sodium hypochlorite only were activated as follows: conventional needle irrigation, ultrasonic activation, sonic activation (EDDY), or laser-based activation (photon-induced photoacoustic streaming/PIPS). For the evaluation of irrigant penetration into dentinal tubules, methylene blue was injected and activated as well. Subsequently, teeth were sectioned horizontally, and dye penetration depths were measured. Alternating sections were split in halves and randomly selected for scanning electron microscopic analysis. Root canal dentine was assessed for smear layer removal and surface disintegration according to a defined scoring system. The data were analyzed statistically with nonparametric and chi-squared tests for whole teeth and separately for coronal, middle, and apical thirds. All the tested activation methods removed a thicker smear layer than needle irrigation only. Additional activation of EDTA improved penetration depths of the irrigants, but not the smear layer removal. Surface disintegration of root canal dentine was observed with the additional activation of EDTA and particularly after laser-based techniques. Additional activation of EDTA does not seem to offer any convincing advantages in terms of irrigant penetration or smear layer removal but disrupts the dentine surface. Especially laser-based activation resulted in undesirable destruction of root canal wall dentine.

Cell homing for dental pulp tissue engineering has been advocated as a feasible approach to regenerate dental pulp in a clinical setting. In order to develop a translational protocol for clinical application, we wanted to determine the effects of disinfectants on the availability of growth factors from the root canal, the amount that can be obtained in this context, and whether they can be processed for use in tissue engineering procedures. The extraction of growth factors should also be confirmed in a clinical setting. Root canals were prepared in 36 extracted mature teeth, and the amount of TGF-β1 in solution was quantified after different irrigation protocols (sodium hypochlorite, chlorhexidine) and after intracanal medication (calcium hydroxide). Centrifugal filters with a cut-off of 10,000 Da and 3000 Da were used for efficient concentration, and volumes and amounts of retained TGF-β1 were measured at different time points. During conventional endodontic treatment, ethylenediaminotetraacetic acid (EDTA) solution was collected after ultrasonic activation from the root canals of mature teeth of 38 patients, and growth factor content was quantified via enzyme-linked immunosorbent assay (ELISA). Irrigation with sodium hypochlorite reduced TGF-β1 release into EDTA. This effect was partially reversed by canal enlargement after the use of sodium hypochlorite and by subsequent use of calcium hydroxide. A few minutes of centrifugation with a cut-off of 10,000 Da reduced the initial volume of the irrigant by 90% and led to a continuous increase in concentration to the same extent. Furthermore, TGF-β1 was obtained from root canals of mature teeth during endodontic treatment in quantities that have been shown to elicit desirable cellular responses in a subsequent clinical application. A mixture with a suitable scaffold material and injection into the root canal has the potential to promote dental pulp regeneration.

Both the dental pulp and the apical papilla represent a promising source of mesenchymal stem cells for regenerative endodontic protocols. The aim of this study was to outline molecular biological conformities and differences between dental pulp stem cells (DPSC) and stem cells from the apical papilla (SCAP). Thus, cells were isolated from the pulp and the apical papilla of an extracted molar and analyzed for mesenchymal stem cell markers as well as multi-lineage differentiation. During induced osteogenic differentiation, viability, proliferation, and wound healing assays were performed, and secreted signaling molecules were quantified by enzyme-linked immunosorbent assays (ELISA). Transcriptome-wide gene expression was profiled by microarrays and validated by quantitative reverse transcription PCR (qRT-PCR). Gene regulation was evaluated in the context of culture parameters and functionality. Both cell types expressed mesenchymal stem cell markers and were able to enter various lineages. DPSC and SCAP showed no significant differences in cell viability, proliferation, or migration; however, variations were observed in the profile of secreted molecules. Transcriptome analysis revealed the most significant gene regulation during the differentiation period, and 13 biomarkers were identified whose regulation was essential for both cell types. DPSC and SCAP share many features and their differentiation follows similar patterns. From a molecular biological perspective, both seem to be equally suitable for dental pulp tissue engineering.

The purpose of this study was to evaluate different protocols of enzymatic treatment (collagenase with either protease, trypsin or hyaluronidase) to isolate mature odontoblasts. Primary odontoblasts were obtained from human molars, which was confirmed by histology and scanning electron microscopy. The combination of collagenase with protease appeared most suitable and resulted in higher cell numbers and better integrity of the odontoblast processes, whereas combination with hyaluronidase or trypsin led to truncated processes and detachment of cell patches instead of single cells. However, trypan blue staining after 24 h showed that odontoblasts in culture did not remain viable. Gene expression analysis was possible after mRNA extraction from tissues ex vivo and real-time semi-quantitative PCR revealed increased expression of collagen, nestin, bone sialoprotein and dentin matrix acidic phosphoprotein 1 in the odontoblast layer. Though primary odontoblasts could not be cultivated after isolation, characteristic genes were identified to differentiate odontoblasts from pulp fibroblasts.

Human dentin is not only a composite material of a collagenous matrix and mineral to provide strength and elasticity to teeth, but also a precious reservoir full of bioactive proteins. They are released after demineralization caused by bacterial acids in carious lesions, by decalcifying irrigants or dental materials and they modulate tissue responses in the underlying dental pulp. This work describes a first-time analysis of the proteome of human dentin using a shotgun proteomic approach that combines three different protein fractionation methods. Dentin matrix proteins were extracted by EDTA and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), OFFGEL isoelectric focusing (IEF) or strong cation exchange chromatography (SCX). Liquid chromatography tandem mass spectrometry (LC-MS/MS) identified 813 human proteins with high confidence, however, isoelectric focusing turned out to be the most beneficial prefractionation method. All Proteins were categorized based on the PANTHER system and representation analysis revealed 31 classes and subclasses to be overrepresented. The acquired knowledge provides a comprehensive insight into the number of proteins in human dentin as well as their physiological and pathological functions. Thus, the data presented paves the way to the analysis of specific functions of dentin matrix proteins in vivo and their potential in tissue engineering approaches to regenerate dental pulp.