Investigation of the early apical release from endodontic hydrogels: A 3D printed model.

AIM: Regenerative Endodontic Procedures (REPs) using new materials such as hydrogels aim to replace current endodontic treatments, but numerous limitations are to overcome. Apical release was little explored in previous studies, especially regarding hydrogels that incorporate molecules, such as growth factors and antibiotics. Apical release is a key mechanism in achieving regeneration, as it could regulate disinfection or cell colonization. Few models exist for apical release, limiting the transfer of these devices from bench to bedside. This study aims to design a simple and standardized model to identify parameters that influence the early apical release kinetic of molecules from endodontic hydrogels. METHODOLOGY: Endodontic Release Inserts (ERI) were designed to mimic the situation of an immature incisor using three different diameters (Ø 0.5 to 2 mm) and to allow the study of the early release from a hydrogel in a 96-well plate. ERI was produced with a 3D printing machine. The kinetic release was investigated using 2 fluorescent, hydrophobic (BDP-500) and hydrophilic (Fluorescein) molecules, in different hydrogels (fibrin and agarose) and in various media (PBS or serum). The release kinetics were estimated by measuring the fluorescence at different time points (1 to 24 h). RESULTS: ERI use made it possible to report that apical diameters increase from 500 to 1000 µm was associated with an increase in release from 4.02 ± 1.63% to 11.53 ± 2.38% over 24 h. It also allowed us to report that bottom solution composition change from PBS to human serum was associated with an increase in the release of fatty acid molecules, whilst a decrease in the hydrogel concentration was associated with a variation in release kinetics. Moreover, nano-encapsulation of a molecule was associated with a decreased release over the first 24 h from 5.25 to 0%. CONCLUSION: ERI use enables investigation of the parameters influencing release kinetics from endodontic hydrogels. Further investigations are necessary to evaluate the interaction of these parameters with each other, in animal models and clinic.

Bioactive Endodontic Hydrogels: From Parameters to Personalized Medicine.

Regenerative endodontic procedures (REPs) aim at recreating dental pulp tissue using biomaterials such as hydrogels. Their bioactivity is mostly related to the nature of biomolecules or chemical compounds that compose the endodontic hydrogel. However, many other parameters, such as hydrogel concentration, bioactive molecules solubility, and apex size, were reported to influence the reciprocal host-biomaterial relationship and hydrogel behavior. The lack of knowledge regarding these various parameters, which should be considered, leads to the inability to predict the clinical outcome and suggests that the biological activity of endodontic hydrogel is impossible to anticipate and could hinder the bench-to-bedside transition. We describe, in this review, that most of these parameters could be identified, described, and studied. A second part of the review lists some challenges and perspectives, including development of future mathematical models that are able to explain, and eventually predict, the bioactivity of endodontic hydrogel used in a clinical setting.

Real-time simulation of the transplanted tooth using model order reduction.

The biomechanics of transplanted teeth remain poorly understood due to a lack of models. In this context, finite element (FE) analysis has been used to evaluate the influence of occlusal morphology and root form on the biomechanical behavior of the transplanted tooth, but the construction of a FE model is extremely time-consuming. Model order reduction (MOR) techniques have been used in the medical field to reduce computing time, and the present study aimed to develop a reduced model of a transplanted tooth using the higher-order proper generalized decomposition method. The FE model of a previous study was used to learn von Mises root stress, and axial and lateral forces were used to simulate different occlusions between 75 and 175N. The error of the reduced model varied between 0.1% and 5.9% according to the subdomain, and was the highest for the highest lateral forces. The time for the FE simulation varied between 2.3 and 7.2 h. In comparison, the reduced model was built in 17s and interpolation of new results took approximately 2.10-2s. The use of MOR reduced the time for delivering the root stresses by a mean 5.9 h. The biomechanical behavior of a transplanted tooth simulated by FE models was accurately captured with a significant decrease of computing time. Future studies could include using jaw tracking devices for clinical use and the development of more realistic real-time simulations of tooth autotransplantation surgery.

Next generation antibacterial strategies for regenerative endodontic procedures: A scoping review.

BACKGROUND: The clinical results following regenerative endodontic procedures (REPs) vary according to numerous parameters, including the presence of bacteria. This limitation reduces the indications for REPs and calls for the development of next generation antibacterial strategies (NGAS) providing alternatives to current antibacterial strategies (CAS) such as double or triple antibiotic paste (DAP/TAP) and (Ca(OH)2). OBJECTIVES: The present scoping review aims to describe the current trends regarding the use of such strategies and highlight future perspectives. METHODS: Four databases (PUBMed, Cochrane, ClinicalTrials and Science Direct) were searched until 1st May 2023. RESULTS: A total of 918 records were identified, 133 were screened and assessed for eligibility, and 87 articles were included. The findings show that (1) clinical studies are only available for CAS, (2) although next generation strategies are the most studied approach since 2017, they are all at the pre-clinical stage, (3) most of the next generation strategies use galenic forms which offer cell support and colonization and which simultaneously contain antibacterial molecules as alternatives to CAS and to antibiotics in general, (4) standardization is required for future research, specifically regarding the bacterial strains studied, the use of biofilm studies and the cellular behaviour assessments. CONCLUSION: Although NGAS are promising strategies to improve REPs in the context of infection, the current evidence is mostly limited to pre-clinical studies. Further methodological improvement is required to allow relevant comparisons between studies and to reduce the time from bench to bedside.

Multifactorial Analysis of Endodontic Microsurgery Using Finite Element Models.

BACKGROUND: The present study aimed to classify the relative contributions of four biomechanical factors-the root-end filling material, the apical preparation, the root resection length, and the bone height-on the root stresses of the resected premolar. METHODS: A design of experiments approach based on a defined subset of factor combinations was conducted to calculate the influence of each factor and their interactions. Sixteen finite element models were created and analyzed using the von Mises stress criterion. The robustness of the design of experiments was evaluated with nine supplementary models. RESULTS: The current study showed that the factors preparation and bone height had a high influence on root stresses. However, it also revealed that nearly half of the biomechanical impact was missed without considering interactions between factors, particularly between resection and preparation. CONCLUSIONS: Design of experiments appears to be a valuable strategy to classify the contributions of biomechanical factors related to endodontics. Imagining all possible interactions and their clinical impact is difficult and can require relying on one's own experience. This study proposed a statistical method to quantify the mechanical risk when planning apicoectomy. A perspective could be to integrate the equation defined herein in future software to support decision-making.

A critical analysis of research methods and experimental models to study pulpitis.

Pulpitis is the inflammatory response of the dental pulp to a tooth insult, whether it is microbial, chemical, or physical in origin. It is traditionally referred to as reversible or irreversible, a classification for therapeutic purposes that determines the capability of the pulp to heal. Recently, new knowledge about dental pulp physiopathology led to orientate therapeutics towards more frequent preservation of pulp vitality. However, full adoption of these vital pulp therapies by dental practitioners will be achieved only following better understanding of cell and tissue mechanisms involved in pulpitis. The current narrative review aimed to discuss the contribution of the most significant experimental models developed to study pulpitis. Traditionally, in vitro two (2D)- or three (3D)-dimensional cell cultures or in vivo animal models were used to analyse the pulp response to pulpitis inducers at cell, tissue or organ level. In vitro, 2D cell cultures were mainly used to decipher the specific roles of key actors of pulp inflammation such as bacterial by-products, pro-inflammatory cytokines, odontoblasts or pulp stem cells. However, these simple models did not reproduce the 3D organisation of the pulp tissue and, with rare exceptions, did not consider interactions between resident cell types. In vitro, tissue/organ-based models were developed to better reflect the complexity of the pulp structure. Their major disadvantage is that they did not allow the analysis of blood supply and innervation participation. On the contrary, in vivo models have allowed researchers to identify key immune, vascular and nervous actors of pulpitis and to understand their function and interplay in the inflamed pulp. However, inflammation was mainly induced by iatrogenic dentine drilling associated with simple pulp exposure to the oral environment or stimulation by individual bacterial by-products for short periods. Clearly, these models did not reflect the long and progressive development of dental caries. Lastly, the substantial diversity of the existing models makes experimental data extrapolation to the clinical situation complicated. Therefore, improvement in the design and standardisation of future models, for example by using novel molecular biomarkers, databased models and artificial intelligence, will be an essential step in building an incremental knowledge of pulpitis in the future.

Validated Finite Element Models of Premolars: A Scoping Review.

Finite element (FE) models are widely used to investigate the biomechanics of reconstructed premolars. However, parameter identification is a complex step because experimental validation cannot always be conducted. The aim of this study was to collect the experimentally validated FE models of premolars, extract their parameters, and discuss trends. A systematic review was performed following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Records were identified in three electronic databases (MEDLINE [PubMed], Scopus, The Cochrane Library) by two independent reviewers. Twenty-seven parameters dealing with failure criteria, model construction, material laws, boundary conditions, and model validation were extracted from the included articles. From 1306 records, 214 were selected for eligibility and entirely read. Among them, 19 studies were included. A heterogeneity was observed for several parameters associated with failure criteria and model construction. Elasticity, linearity, and isotropy were more often chosen for dental and periodontal tissues with a Young's modulus mostly set at 18-18.6 GPa for dentine. Loading was mainly simulated by an axial force, and FE models were mostly validated by in vitro tests evaluating tooth strains, but different conditions about experiment type, sample size, and tooth status (intact or restored) were reported. In conclusion, material laws identified herein could be applied to future premolar FE models. However, further investigations such as sensitivity analysis are required for several parameters to clarify their indication.

Comparison of Two Innovative Strategies Using Augmented Reality for Communication in Aesthetic Dentistry: A Pilot Study.

During dental prosthetic rehabilitation, communication and conception are achieved using rigorous methodologies such as smile design protocols. The aim of the present pilot study was to compare two innovative strategies that used augmented reality for communication in dentistry. These strategies enable the user to instantly try a virtual smile proposition by taking a set of pictures from different points of view or by using the iPad as an enhanced mirror. Sixth-year dental students (n=18, women = 13, men = 5, mean age = 23.8) were included in this pilot study and were asked to answer a 5-question questionnaire studying the user experience using a visual analog scale (VAS). Answers were converted into a numerical result ranging from 0 to 100 for statistical analysis. Participants were not able to report a difference between the two strategies in terms of handling of the device (p=0.45), quality of the reconstruction (p=0.73), and fluidity of the software (p=0.67). Even if the participants' experience with the enhanced mirror was more often reported as immersive and more likely to be integrated in a daily dental office practice, no significant increase was reported (p=0.15 and p=0.07). Further investigations are required to evaluate time and cost savings in daily practice. Software accuracy is also a major point to investigate in order to go further in clinical applications.

Phenotypic Identification of Dental Pulp Mesenchymal Stem/Stromal Cells Subpopulations with Multiparametric Flow Cytometry.

Dental pulp (DP) is a specialized, highly vascularized, and innervated connective tissue mainly composed of undifferentiated mesenchymal cells, fibroblasts, and highly differentiated dentin-forming odontoblasts. Undifferentiated mesenchymal cells include stem/stromal cell populations usually called dental pulp mesenchymal stem cells (DP-MSCs) which differ in their self-renewal properties, lineage commitment, and differentiation capabilities. Analysis of surface antigens has been largely used to precisely identify these DP-MSC populations. However, a major difficulty is that these antigens are actually not specific for MSCs. Most of the markers used are indeed shared by other cell populations such as progenitor cells, mature fibroblasts, and/or perivascular cells. Accordingly, the detection of only one of these markers in a cell population is clearly insufficient to determine its stemness. Recent data reported that multiparametric flow cytometry, by allowing for the detection of several molecules on the surface of one single cell, is a powerful tool to elucidate the phenotype of a cell population both in vivo and in vitro. So far, DP-MSC populations have been characterized mainly based on the isolated expression of molecules known to be expressed by stem cells, such as Stro-1 antigen, melanoma cell adhesion molecule MCAM/CD146, low-affinity nerve growth factor receptor p75NTR/CD271, and the mesenchymal stem cell antigen MSCA-1. Using multiparametric flow cytometry, we recently showed that human DP-MSCs are indeed phenotypically heterogeneous and form several populations.The present paper describes the multiparametric flow cytometry protocol we routinely use for characterizing DP-MSCs. The description includes the design of the antibody panel and explains the selection of the different parameters related to the data quality control.

Design and characterization of a chitosan-enriched fibrin hydrogel for human dental pulp regeneration.

OBJECTIVE: Regenerating a functional dental pulp in the pulpectomized root canal has been recently proposed as a novel therapeutic strategy in dentistry. To reach this goal, designing an appropriate scaffold able to prevent the growth of residual endodontic bacteria, while supporting dental pulp tissue neoformation, is needed. Our aim was to create an innovative cellularized fibrin hydrogel supplemented with chitosan to confer this hydrogel antibacterial property. METHODS: Several fibrin-chitosan formulations were first screened by rheological analyses, and the most appropriate for clinical use was then studied in terms of microstructure (by scanning electron microscopy), antimicrobial effect (analysis of Enterococcus fæcalis growth), dental pulp-mesenchymal stem/stromal cell (DP-MSC) viability and spreading after 7 days of culture (LiveDead® test), DP-MSC ultrastructure and extracellular matrix deposition (transmission electron microscopy), and DP-MSC proliferation and collagen production (RT-qPCR and immunohistochemistry). RESULTS: A formulation associating 10mg/mL fibrinogen and 0.5% (w/w), 40% degree of acetylation, medium molar mass chitosan was found to be relevant in order to forming a fibrin-chitosan hydrogel at cytocompatible pH (# 7.2). Comparative analysis of fibrin-alone and fibrin-chitosan hydrogels revealed a potent antibacterial effect of the chitosan in the fibrin network, and similar DP-MSC viability, fibroblast-like morphology, proliferation rate and type I/III collagen production capacity. SIGNIFICANCE: These results indicate that incorporating chitosan within a fibrin hydrogel would be beneficial to promote human DP tissue neoformation thanks to chitosan antibacterial effect and the absence of significant detrimental effect of chitosan on dental pulp cell morphology, viability, proliferation and collagenous matrix production.

Multi-Fiber-Reinforced Composites for the Coronoradicular Reconstruction of Premolar Teeth: A Finite Element Analysis.

A coronoradicular reconstruction (CRR) has conventionally used a metallic inlay core (MIC) or a single-fiber-reinforced composite (sFRC) but extensive dentin removal can lead to root fracture. We propose herein a multi-fiber-reinforced composite (mFRC) based on a bundle of thin flexible fibers that can be adapted to the root anatomy without removing additional dentin. The aim of this study was to compare the mechanical behavior of the root reconstructed with mFRC, MIC, or sFRC using a finite element analysis (FEA). Models with or without a ferrule effect were created using Autodesk© software and divided into four parts: root, post, bonding composite or cement, and zirconia crown. For both models, extreme stress values (ESV), stress distribution, and risk of fracture were calculated for an oblique force (45°) of 100 N applied to the top of the buccal cusp. Results indicated that mFRC and mFRCG present a lower risk of fracture of the root and of the CRR without ferrule and thus could be valuable alternatives for premolar CRR. Further studies are necessary to evaluate the clinical success of these CRR.

Manufacturing of an immediate removable partial denture with an intraoral scanner and CAD-CAM technology: a case report.

BACKGROUND: Incisor loss constitutes a strong aesthetic and psychologic traumatism for the patient and it remains a challenging situation for the dental practitioner because of the necessity to rapidly replace the lacking tooth. Various therapeutic procedures have been proposed to replace the incisor concerned, for example by using a removable partial denture. However, the manufacturing of such a denture with classical procedures is often subject to processing errors and inaccuracies. The computer-aided design and computer-aided manufacturing (CAD-CAM) technology could represent a good alternative, but it is currently difficult because of the lack of dental softwares able to design easily immediate removable partial dentures. CASE PRESENTATION: A 30-year- patient complained about pain caused by a horizontally and vertically mobile maxillary right central incisor. After all options were presented, extraction of the traumatized incisor was decided due to its very poor prognosis, and the patient selected the realization of a removable denture for economic reasons. The present paper proposes an innovative procedure for immediate removable denture, based on the use of an intraoral scanner, CAD with two different softwares used sequentially, and CAM with a 5-axis machine. CONCLUSIONS: We show in this report that associating an intraoral scanner and CAD-CAM technology can be extended to immediate dentures manufacturing, which could be a valuable procedure for dental practitioners and laboratories, and also for patients.

Intraoral Scanner Technologies: A Review to Make a Successful Impression.

To overcome difficulties associated with conventional techniques, impressions with IOS (intraoral scanner) and CAD/CAM (computer-aided design and manufacturing) technologies were developed for dental practice. The last decade has seen an increasing number of optical IOS devices, and these are based on different technologies; the choice of which may impact on clinical use. To allow informed choice before purchasing or renewing an IOS, this article summarizes first the technologies currently used (light projection, distance object determination, and reconstruction). In the second section, the clinical considerations of each strategy such as handling, learning curve, powdering, scanning paths, tracking, and mesh quality are discussed. The last section is dedicated to the accuracy of files and of the intermaxillary relationship registered with IOS as the rendering of files in the graphical user interface is often misleading. This overview leads to the conclusion that the current IOS is adapted for a common practice, although differences exist between the technologies employed. An important aspect highlighted in this review is the reduction in the volume of hardware which has led to an increase in the importance of software-based technologies.

Current challenges in human tooth revitalization.

Tooth vitality and health are related to the presence of a living connective tissue, the dental pulp (DP), in the center of the dental organ. The DP contains the tooth immune defence system that is activated against invading oral cariogenic bacteria during the caries process and the tissue repair/regeneration machinery involved following microorganisms' eradication. However, penetration of oral bacteria into the DP often leads to complete tissue destruction and colonization of the endodontic space by microorganisms. Classical endodontic therapies consist of disinfecting then sealing the endodontic space with a gutta percha-based material. However, re-infections of the endodontic space by oral bacteria can occur, owing to the lack of tightness of the material. Recent findings suggest that regenerating a fully functional pulp tissue may be an ideal therapeutic solution to maintain a tooth defence system that will detect and help manage future injuries. The objective of this paper was to explain the different revascularization and regeneration strategies that have been proposed to reconstitute a living DP tissue and to discuss the main challenges that have to be resolved to improve these therapeutic strategies.

Immunophenotyping Reveals the Diversity of Human Dental Pulp Mesenchymal Stromal Cells In vivo and Their Evolution upon In vitro Amplification.

Mesenchymal stromal/stem cells (MSCs) from human dental pulp (DP) can be expanded in vitro for cell-based and regenerative dentistry therapeutic purposes. However, their heterogeneity may be a hurdle to the achievement of reproducible and predictable therapeutic outcomes. To get a better knowledge about this heterogeneity, we designed a flow cytometric strategy to analyze the phenotype of DP cells in vivo and upon in vitro expansion with stem cell markers. We focused on the CD31- cell population to exclude endothelial and leukocytic cells. Results showed that the in vivo CD31- DP cell population contained 1.4% of CD56+, 1.5% of CD146+, 2.4% of CD271+ and 6.3% of MSCA-1+ cells but very few Stro-1+ cells (≤ 1%). CD56+, CD146+, CD271+, and MSCA-1+ cell subpopulations expressed various levels of these markers. CD146+MSCA-1+, CD271+MSCA-1+, and CD146+CD271+ cells were the most abundant DP-MSC populations. Analysis of DP-MSCs expanded in vitro with a medicinal manufacturing approach showed that CD146 was expressed by about 50% of CD56+, CD271+, MSCA-1+, and Stro-1+ cells, and MSCA-1 by 15-30% of CD56+, CD146+, CD271+, and Stro-1+ cells. These ratios remained stable with passages. CD271 and Stro-1 were expressed by <1% of the expanded cell populations. Interestingly, the percentage of CD56+ cells strongly increased from P1 (25%) to P4 (80%) both in all sub-populations studied. CD146+CD56+, MSCA-1+CD56+, and CD146+MSCA-1+ cells were the most abundant DP-MSCs at the end of P4. These results established that DP-MSCs constitute a heterogeneous mixture of cells in pulp tissue in vivo and in culture, and that their phenotype is modified upon in vitro expansion. Further studies are needed to determine whether co-expression of specific MSC markers confers DP cells specific properties that could be used for the regeneration of human tissues, including the dental pulp, with standardized cell-based medicinal products.

Dental Pulp Defence and Repair Mechanisms in Dental Caries.

Dental caries is a chronic infectious disease resulting from the penetration of oral bacteria into the enamel and dentin. Microorganisms subsequently trigger inflammatory responses in the dental pulp. These events can lead to pulp healing if the infection is not too severe following the removal of diseased enamel and dentin tissues and clinical restoration of the tooth. However, chronic inflammation often persists in the pulp despite treatment, inducing permanent loss of normal tissue and reducing innate repair capacities. For complete tooth healing the formation of a reactionary/reparative dentin barrier to distance and protect the pulp from infectious agents and restorative materials is required. Clinical and in vitro experimental data clearly indicate that dentin barrier formation only occurs when pulp inflammation and infection are minimised, thus enabling reestablishment of tissue homeostasis and health. Therefore, promoting the resolution of pulp inflammation may provide a valuable therapeutic opportunity to ensure the sustainability of dental treatments. This paper focusses on key cellular and molecular mechanisms involved in pulp responses to bacteria and in the pulpal transition between caries-induced inflammation and dentinogenic-based repair. We report, using selected examples, different strategies potentially used by odontoblasts and specialized immune cells to combat dentin-invading bacteria in vivo.

[Innovations in diagnosis and treatment about a case of primary failure eruption linked to a PTHR1 gene mutation]. / Innovations dans le diagnostic et le traitement d'un cas de défaut primaire d'éruption lié à une mutation du gène PTHR1.

Primary failure of eruption is a rare condition marked by non-eruption of the posterior teeth due to mutation of a gene responsible for tooth eruption. Today, this anomaly can be detected early using innovative 3D-imaging techniques. Genetic and histologic testing will confirm the diagnosis and unfavorable prognosis. Alveolar growth must be followed in other areas too in order to avoid structural and functional asymmetry. An analysis of the diagnostic and therapeutic options using bone-borne anchorage is presented via the long-term monitoring of a female patient presenting primary failure of eruption linked to mutation of the PTHR1 gene.

Manufacturing of dental pulp cell-based products from human third molars: current strategies and future investigations.

In recent years, mesenchymal cell-based products have been developed to improve surgical therapies aimed at repairing human tissues. In this context, the tooth has recently emerged as a valuable source of stem/progenitor cells for regenerating orofacial tissues, with easy access to pulp tissue and high differentiation potential of dental pulp mesenchymal cells. International guidelines now recommend the use of standardized procedures for cell isolation, storage and expansion in culture to ensure optimal reproducibility, efficacy and safety when cells are used for clinical application. However, most dental pulp cell-based medicinal products manufacturing procedures may not be fully satisfactory since they could alter the cells biological properties and the quality of derived products. Cell isolation, enrichment and cryopreservation procedures combined to long-term expansion in culture media containing xeno- and allogeneic components are known to affect cell phenotype, viability, proliferation and differentiation capacities. This article focuses on current manufacturing strategies of dental pulp cell-based medicinal products and proposes a new protocol to improve efficiency, reproducibility and safety of these strategies.

Production of Human Dental Pulp Cells with a Medicinal Manufacturing Approach.

INTRODUCTION: Human dental pulp cells (HDPCs) are generally isolated and cultured with xenogeneic products and in stress conditions that may alter their biological features. However, guidelines from the American Food and Drug Administration and the European Medicines Agency currently recommend the use of protocols compliant with medicinal manufacturing. Our aim was to design an ex vivo procedure to produce large amounts of HDPCs for dentin/pulp and bone engineering according to these international recommendations. METHODS: HDPC isolation was performed from pulp explant cultures. After appropriate serum-free medium selection, cultured HDPCs were immunophenotyped with flow cytometry. Samples were then cryopreserved for 510 days. The post-thaw cell doubling time was determined up to passage 4 (P4). Karyotyping was performed by G-band analysis. Osteo/odontoblastic differentiation capability was determined after culture in a differentiation medium by gene expression analysis of osteo/odontoblast markers and mineralization quantification. RESULTS: Immunophenotyping of cultured HDPCs revealed a mesenchymal profile of the cells, some of which also expressed the stem/progenitor cell markers CD271, Stro-1, CD146, or MSCA-1. The post-thaw cell doubling times were stable and similar to fresh HDPCs. Cells displayed no karyotype abnormality. Alkaline phosphatase, osteocalcin, and dentin sialophosphoprotein gene expression and culture mineralization were increased in post-thaw HDPC cultures performed in differentiation medium compared with cultures in control medium. CONCLUSIONS: We successfully isolated, cryopreserved, and amplified human dental pulp cells with a medicinal manufacturing approach. These findings may constitute a basis on which to investigate how HDPC production can be optimized for human pulp/dentin and bone tissue engineering.

Human odontoblast-like cells produce nitric oxide with antibacterial activity upon TLR2 activation.

The penetration of cariogenic oral bacteria into enamel and dentin during the caries process triggers an immune/inflammatory response in the underlying pulp tissue, the reduction of which is considered a prerequisite to dentinogenesis-based pulp regeneration. If the role of odontoblasts in dentin formation is well known, their involvement in the antibacterial response of the dental pulp to cariogenic microorganisms has yet to be elucidated. Our aim here was to determine if odontoblasts produce nitric oxide (NO) with antibacterial activity upon activation of Toll-like receptor-2 (TLR2), a cell membrane receptor involved in the recognition of cariogenic Gram-positive bacteria. Human odontoblast-like cells differentiated from dental pulp explants were stimulated with the TLR2 synthetic agonist Pam2CSK4. We found that NOS1, NOS2, and NOS3 gene expression was increased in Pam2CSK4-stimulated odontoblast-like cells compared to unstimulated ones. NOS2 was the most up-regulated gene. NOS1 and NOS3 proteins were not detected in Pam2CSK4-stimulated or control cultures. NOS2 protein synthesis, NOS activity and NO extracellular release were all augmented in stimulated samples. Pam2CSK4-stimulated cell supernatants reduced Streptococcus mutans growth, an effect counteracted by the NOS inhibitor L-NAME. In vivo, the NOS2 gene was up-regulated in the inflamed pulp of carious teeth compared with healthy ones. NOS2 protein was immunolocalized in odontoblasts situated beneath the caries lesion but not in pulp cells from healthy teeth. These results suggest that odontoblasts may participate to the antimicrobial pulp response to dentin-invading Gram-positive bacteria through NOS2-mediated NO production. They might in this manner pave the way for accurate dental pulp healing and regeneration.