European Society of Endodontology position statement: endodontic management of traumatized permanent teeth.

This position statement represents a consensus of an expert committee convened by the European Society of Endodontology (ESE) on the endodontic management of traumatized permanent teeth. A recent comprehensive review with detailed background information provides the basis for this position statement (Krastl et al. 2021, International Endodontic Journal, https://doi.org/10.1111/iej.13508). The statement is based on current scientific evidence as well as the expertise of the committee. Complementing the recently revised guidelines of the International Association of Dental Traumatology, this position statement aims to provide clinical guidance for the choice of the appropriate endodontic approach for traumatized permanent teeth. Given the dynamic nature of research in this area, this position statement will be updated at appropriate intervals.

Histology of human teeth: Standard and specific staining methods revisited.

OBJECTIVE: Histological techniques have long been an integral part of dental research. Especially the processing of complex tissues poses specific challenges, however, literature offers only few technical references. Objectives of this study were therefore to optimize histological staining methods and compile detailed protocols for preparation and staining of dental tissues. METHODS: Human teeth were collected and fixed with 4 % formaldehyde solution after extraction. Subsequently, teeth were decalcified in 17 % EDTA or Morse's solution over a period of 28 days. The extent of decalcification was determined by weight loss and radiography. After sectioning, histological staining methods were optimized for their use on teeth. These included hematoxylin-eosin, Masson trichrome, Masson-Goldner trichrome and May-Gruenwald-Giemsa staining. Nerve fibres were visualized by luxol fast blue staining and Bodian silver staining. In addition, specific methods like TRAP, modified Brown and Brenn as well as picrosirius red staining with light polarization or fluorescence were applied and optimized. RESULTS: Preparation of an artificial access to the pulp chamber was essential to ensure prompt penetration of the chemicals. Decalcification with Morse's solution took at least two weeks but was more efficient than 17 % ETDA, where thorough demineralization was achieved only after three weeks. The staining methods exhibited differences not only regarding their ability to display specific structures of interest, but also in terms of reproducibility. CONCLUSION: High-quality histology of teeth can only be achieved after optimal tissue preparation and accurate staining. A complementary use of staining techniques is necessary to answer specific research questions.

Endodontic management of traumatized permanent teeth: a comprehensive review.

The pulp plays a key role in the treatment of traumatic dental injuries (TDIs) and is strongly associated with the outcome, particularly in severe cases. A correct pulp diagnosis is essential as it forms the basis for developing the appropriate management strategy. However, many TDIs are complex, and their treatment requires a profound knowledge of the physiological and pathological responses of the affected tissues. This comprehensive review will look at the dentine-pulp complex and its interaction with the surrounding tissues following TDIs. The literature up to 2020 was reviewed based on several searches on PubMed and the Cochrane Library using relevant terms. In addition to the recently revised guidelines of the International Association of Dental Traumatology, this article aims to provide background information with a focus on endodontic aspects and to gather evidence on which a clinician can make decisions on the choice of the appropriate endodontic approach for traumatized permanent teeth.

Biomechanical performance of an immature maxillary central incisor after revitalization: a finite element analysis.

AIM: To investigate the stress distribution in an immature maxillary incisor and the same tooth after simulated revitalization with deposition of tubular dentine or cementum by finite element analysis (FEA). METHODOLOGY: A finite element model of a maxillary central incisor was developed on the basis of a µCT scan. The tooth was segmented in two parts: a part that represented a tooth in an immature state and an apical part that represented the tissue formed after revitalization. The apical part was given the mechanical properties of dentine or cementum. The immature tooth and the same tooth reinforced by either dentine or cementum underwent simulation of biting, trauma and orthodontic movement. Von Mises stress values were compared between the scenarios and tooth segments. RESULTS: Maximum stress in the immature incisor developed apically; however, dentine- and cementum-reinforced teeth revealed the greatest stress in the external portion of the root decreasing towards the apex. Greatest mechanical stress was caused by dental trauma perpendicular to the long axis of the root followed by biting and orthodontic movement. Stress peaks were lower in the dentine-reinforced tooth compared with the cementum-reinforced tooth in all scenarios; however, median stress in the immature part was reduced irrespective of dentine or cementum deposition. Dentine reinforcement caused greater stress values in the apical segment due to absorbance of the applied force, whereas stress was not transferred towards deposited cementum. CONCLUSIONS: Apposition of simulated hard tissue in a maxillary central incisor after revitalization reduced mechanical stress in the immature tooth. Formation of dentine was advantageous because, unlike cementum, it facilitated an even stress distribution throughout the root resulting in lower stress values.

Penetration depth of irrigants into root dentine after sonic, ultrasonic and photoacoustic activation.

AIM: To compare penetration depths of endodontic irrigants into the dentinal tubules of extracted teeth when using several activation methods. METHODOLOGY: The root canals of 90 extracted human teeth were prepared to size 40, .06 taper. The straight and round-shaped root canals were distributed randomly into six groups, and final irrigation was performed with EDTA and sodium hypochlorite as follows: (I) manual dynamic activation, (II) Ultrasonic, (III) Sonic, (IV) PIPS (photon-induced photoacoustic streaming, (V) SWEEPS (shock-wave enhanced emission photoacoustic streaming) and (0) control without final irrigation or activation. Subsequently, methylene blue was inserted into the canals and activated according to the groups (I-V). Teeth were sectioned horizontally, imaged under a light microscope, and dye penetration depths were measured in six sections per tooth and 24 points on a virtual clock-face per section. Data were analysed statistically by nonparametric tests for whole teeth and separately for coronal, middle and apical thirds. RESULTS: Penetration of dye into the dentinal tubules was lowest for the controls. Median penetration depths amounted to 700-900 µm for groups I-V with differences in the apical thirds between group I and the other test groups. Minimum penetration depths were significantly greater for PIPS in the apical thirds (P ≤ 0.046). CONCLUSIONS: Greater penetration depths occurred in the apical thirds for ultrasonic, sonic and laser-induced activation compared to manual dynamic activation. PIPS was associated with deeper penetration of irrigants. The novel SWEEPS mode did not increase irrigant penetration.

European Society of Endodontology position statement: Management of deep caries and the exposed pulp.

This position statement on the management of deep caries and the exposed pulp represents the consensus of an expert committee, convened by the European Society of Endodontology (ESE). Preserving the pulp in a healthy state with sustained vitality, preventing apical periodontitis and developing minimally invasive biologically based therapies are key themes within contemporary clinical endodontics. The aim of this statement was to summarize current best evidence on the diagnosis and classification of deep caries and caries-induced pulpal disease, as well as indicating appropriate clinical management strategies for avoiding and treating pulp exposure in permanent teeth with deep or extremely deep caries. In presenting these findings, areas of controversy, low-quality evidence and uncertainties are highlighted, prior to recommendations for each area of interest. A recently published review article provides more detailed information and was the basis for this position statement (Bjørndal et al. 2019, International Endodontic Journal, doi:10.1111/iej.13128). The intention of this position statement is to provide the practitioner with relevant clinical guidance in this rapidly developing area. An update will be provided within 5 years as further evidence emerges.

Interactive effects of LPS and dentine matrix proteins on human dental pulp stem cells.

AIM: To investigate the combinatorial effects of lipopolysaccharide (LPS) and extracted dentine matrix proteins (eDMP) on regenerative and inflammatory responses in human dental pulp stem cells (DPSCs). METHODOLOGY: Culture media were supplemented with several concentrations of LPS, eDMP and combinations of both. Cell viability was assessed over 1 week by MTT assay; cell survival was evaluated after 24 h and 7 days by flow cytometry. The expression of mineralization-associated marker genes was determined by real-time quantitative polymerase chain reaction (RT-qPCR). To analyse the inflammatory response, secretion of interleukin 6 (IL-6) was quantified in the initial and the late phase of cell culture by enzyme-linked immunosorbent assay (ELISA). Data were treated nonparametrically and Mann-Whitney U-tests were performed to compare all experimental groups (α = 0.05). RESULTS: Whereas LPS had no impact on viability, eDMP led to a concentration-dependent decrease, which was significant after 7 days (P ≤ 0.024). A moderate decline of cell survival induced by LPS was detected after 48 h (P ≤ 0.026), whereas eDMP was able to reverse this effect. eDMP alone caused increased expression of tested marker genes, LPS had no regulatory effect. Combined eDMP and LPS induced an upregulation of collagen type I and osteocalcin, whereas expression levels of dentine matrix acidic phosphoprotein and dentine sialophosphoprotein were similar to the control. IL-6-secretion was increased by LPS over time. eDMP markedly elevated initial production of IL-6 (P ≤ 0.002), but suppressed LPS-induced cytokine production in the later phase. CONCLUSIONS: Lipopolysaccharide did not affect cell viability but interfered with odontoblast-like cell differentiation of DPSCs. Proteins from the dentine matrix may have a protective effect, attenuate the detrimental impact of LPS and thus play an important role during pulp repair.

A training model for revitalization procedures.

AIM: To develop an inexpensive simulation model for training of revitalization procedures. METHODOLOGY: A replica of an immature maxillary central incisor was equipped with a mock blood reservoir at the root apex and embedded in a plaster model. Mock blood consisted of water supplemented with red pigments and fibrinogen, whilst thrombin was inserted into the root canal to allow for clot formation. RESULTS: A true-to-life training model for revitalization procedures was established, where the induction of bleeding and formation of a blood clot can be mimicked. The model can be fastened to a patient mannequin and thus closely simulate the clinical setting. CONCLUSIONS: The newly developed model can be used for training purposes, both for dental students and for practitioners, to perform the treatment steps of a revitalization procedure.

Dentine matrix proteins: isolation and effects on human pulp cells.

AIM: To establish a simplified and efficient protocol for the isolation and concentration of matrix proteins from human dentine, and to assess the effects of extracted dentine matrix proteins (eDMP) on the behaviour of human pulp cells. METHODOLOGY: Matrix proteins were isolated from human dentine, purified, concentrated and characterized with protein and enzyme-linked immunosorbent assays (ELISA). Culture media were supplemented with eDMP in different concentrations, referred to as eDMP 1-10 000, to assess viability and proliferation of human pulp cells by DNA and MTT assays; apoptotic events were quantified by flow cytometry. Chemotactic effects of eDMP were assessed in a modified Boyden chamber assay. Expression levels of odontoblastic marker genes in pulp cells cultured with eDMPs were determined by real-time quantitative PCR, and the ability to induce mineralization was demonstrated by alizarin red staining. Nonparametric statistical analysis was performed to pairwise compare different groups at all time-points (Mann-Whitney U-test, α = 0.05). RESULTS: High concentrations of eDMP exhibited significant antiproliferative effects (P ≤ 0.023) after 5 (eDMP 1000) and 7 days (eDMP 500) without affecting cell viability. Apoptosis was barely influenced (P ≥ 0.089). eDMP exerted a concentration-dependent chemotactic stimulus on dental pulp cells with statistical significance already at low dosage (P = 0.006 at eDMP 10). Changes in gene expression indicated a differentiation into odontoblast-like cells, which was corroborated by findings of mineral nodule formation. CONCLUSIONS: A novel, effective and time-saving protocol for isolation and concentration of dentine matrix proteins is presented. As eDMP stimulates chemotaxis, differentiation and mineralization without affecting viability, endogenous dentine matrix proteins might be valuable for approaches to regenerate or engineer dental pulp.

Ultrasonic activation of irrigants increases growth factor release from human dentine.

OBJECTIVES: Bioactive proteins are sequestered in human dentine and play a decisive role in dental pulp regeneration and repair. They can be released and exposed on the dentine surface by acids, but also chelators, such as ethylenediaminetetraacetic acid (EDTA). The objectives of this study were (i) to evaluate whether ultrasonic activation of irrigants in the root canal will promote growth factor release from dentine and (ii) to collect bioactive proteins in a physiological solution. MATERIALS AND METHODS: Human dentine disks underwent irrigation with and without ultrasonic activation. The protocols included treatment by either a single or two consecutive steps with 10 % EDTA and phosphate-buffered saline (PBS), where each sample was treated three times. To mimic clinical conditions, selected irrigation regimens were applied to root canals of extracted human teeth after preparation. Amounts of transforming growth factor ß1 (TGF-ß1) in solution were quantified using enzyme-linked immunosorbent assays. Nonparametric statistical analysis was performed to compare different groups as well as repetitions within a group (Mann-Whitney U test, α = 0.05). Additionally, morphological changes of dentine surfaces were visualized by scanning electron microscopy (SEM). RESULTS: TGF-ß1 was not detectable after irrigation of dentine with PBS, neither with nor without ultrasonic activation. Irrigation with EDTA released TGF-ß1, and ultrasonic activation of EDTA enhanced this effect. However, preceding EDTA conditioning enabled the release of bioactive proteins into PBS solution. Similar results were observed in dentine disks and root canals. Visualization of dentine surfaces after different treatment revealed superficial erosion after ultrasonic activation irrespective of the irrigant solution, but different degrees of exposure of organic substance. CONCLUSIONS: Ultrasonic activation enhances growth factor release from human dentine. Bioactive proteins can be isolated in physiological solvents and may act as autologous supplements for regenerative endodontic treatment or pulp tissue engineering. CLINICAL RELEVANCE: Autologous growth factors from human dentine can advance treatment strategies in dental pulp tissue engineering.

European Society of Endodontology position statement: Revitalization procedures.

This position statement represents a consensus of an expert committee convened by the European Society of Endodontology (ESE) on revitalization procedures. The statement is based on current clinical and scientific evidence as well as the expertise of the committee. The goal is to provide suitably trained dentists with a protocol including procedural details for the treatment of immature teeth with pulp necrosis as well as a patient consent form. Revitalization is a biologically based treatment as an alternative to apexification in properly selected cases. Previously published review articles provide more detailed background information and the basis for this position statement (Journal of Endodontics, 39, 2013, S30; Journal of Endodontics, 39, 2013, 319; Journal of Endodontics, 40, 2014, 1045; Dental Traumatology, 31, 2015, 267; International Endodontic Journal, 2015, doi: 10.1111/iej.12606). As controlled clinical trials are lacking and new evidence is still emerging, this position statement will be updated at appropriate intervals. This might lead to changes to the protocol provided here.

Clinical procedures for revitalization: current knowledge and considerations.

Revitalization or regenerative treatment approaches in teeth with incomplete root formation and pulp necrosis have become part of the therapeutic endodontic spectrum and should be considered as an alternative to conventional apexification. Ideally, regenerative endodontic procedures allow not only for a resolution of pain, inflammation and periapical lesions, but also for the formation of an immunocompetent tissue inside the root canal which can reconstitute the original biological structure and function of dental pulp and thus lead to an increase in root length, and thickness and strength of previously thin, fracture-prone dentine walls. Common features of regenerative procedures performed in immature teeth with pulp necrosis include (i) minimal or no instrumentation of the dentinal walls, (ii) disinfection with irrigants, (iii) application of an intracanal medicament, (iv) provocation of bleeding into the canal and creation of a blood clot, (v) capping with calcium silicate, and (vi) coronal seal. Although case reports and case series provide promising results, the protocol for regenerative endodontic treatment is not fully established; questions remain regarding the terminology, patient selection and informed consent as well as procedural details, especially on the choice of irrigants, intracanal medicaments and materials for cavity sealing. Animal studies document repair rather than regeneration, which opens the discussion on prognosis and outcome, especially the biological versus the patient-based outcome. This review will provide an overview of the current state of regenerative endodontic therapies, discuss open questions and provide recommendations based on the recent literature.

EDTA conditioning of dentine promotes adhesion, migration and differentiation of dental pulp stem cells.

AIM: To evaluate the effect of dentine conditioning on migration, adhesion and differentiation of dental pulp stem cells. METHODOLOGY: Dentine discs prepared from extracted human molars were pre-treated with EDTA (10%), NaOCl (5.25%) or H2 O. Migration of dental pulp stem cells towards pre-treated dentine after 24 and 48 h was assessed in a modified Boyden chamber assay. Cell adhesion was evaluated indirectly by measuring cell viability. Expression of mineralization-associated genes (COL1A1, ALP, BSP, DSPP, RUNX2) in cells cultured on pre-treated dentine for 7 days was determined by RT-qPCR. Nonparametric statistical analysis was performed for cell migration and cell viability data to compare different groups and time-points (Mann-Whitney U-test, α = 0.05). RESULTS: Treatment of dentine with H2 O or EDTA allowed for cell attachment, which was prohibited by NaOCl with statistical significance (P = 0.000). Furthermore, EDTA conditioning induced cell migration towards dentine. The expression of mineralization-associated genes was increased in dental pulp cells cultured on dentine after EDTA conditioning compared to H2 O-pre-treated dentine discs. CONCLUSIONS: EDTA conditioning of dentine promoted the adhesion, migration and differentiation of dental pulp stem cells towards or onto dentine. A pre-treatment with EDTA as the final step of an irrigation protocol for regenerative endodontic procedures has the potential to act favourably on new tissue formation within the root canal.

Three-dimensional culture of dental pulp stem cells in direct contact to tricalcium silicate cements.

OBJECTIVES: Calcium silicate cements are biocompatible dental materials applicable in contact with vital tissue. The novel tricalcium silicate cement Biodentine™ offers properties superior to commonly used mineral trioxide aggregate (MTA). Objective of this study was to evaluate its cytocompatibility and ability to induce differentiation and mineralization in three-dimensional cultures of dental pulp stem cells after direct contact with the material. MATERIALS AND METHODS: Test materials included a new tricalcium silicate (Biodentine™, Septodont, Saint-Maur-des-Fossés, France), MTA (ProRoot® MTA, DENSPLY Tulsa Dental Specialities, Johnson City, TN, USA), glass ionomer (Ketac™ Molar Aplicap™, 3M ESPE, Seefeld, Germany), human dentin disks and polystyrene. Magnetic activated cell sorting for to the surface antigen STRO-1 was performed to gain a fraction enriched with mesenchymal stem cells. Samples were allowed to set and dental pulp stem cells in collagen carriers were placed on top. Scanning electron microscopy of tricalcium silicate cement surfaces with and without cells was conducted. Cell viability was measured for 14 days by MTT assay. Alkaline phosphatase activity was evaluated (days 3, 7, and 14) and expression of mineralization-associated genes (COL1A1, ALP, DSPP, and RUNX2) was quantified by real-time quantitative PCR. Nonparametric statistical analysis for cell viability and alkaline phosphatase data was performed to compare different materials as well as time points (Mann-Whitney U test, α = 0.05). RESULTS: Cell viability was highest on tricalcium silicate cement, followed by MTA. Viability on glass ionomer cement and dentin disks was significantly lower. Alkaline phosphatase activity was lower in cells on new tricalcium silicate cement compared to MTA, whereas expression patterns of marker genes were alike. CONCLUSIONS: Increased cell viability and similar levels of mineralization-associated gene expression in three-dimensional cell cultures on the novel tricalcium silicate cement and mineral trioxide aggregate indicate that the material is cytocompatible and bioactive. CLINICAL RELEVANCE: The tested new tricalcium silicate cement confirms its suitability as an alternative to MTA in vital pulp therapy.

Material Tissue Interaction--From Toxicity to Tissue Regeneration.

The topic of material tissue interaction has gained increasing interest over recent decades from both the dental profession and the public. The primary goal initially was to avoid adverse reactions after the application of dental materials. New laboratory test methods have been developed, and currently premarket testing programs, which attempt to guarantee a basic level of patient safety, are legally required worldwide. The dentist is responsible for selecting the correct indication as well as the proper handling of any newly emerging risk. Apart from this phenomenon-oriented "inert materials concept," the "analytical concept" focuses primarily on analyzing the reasons for adverse reactions, and identifying their associated modifying factors, in order to prevent them or to develop new and more biocompatible materials. The "concept of bioactivity" involves addressing the possibility of positively influencing tissue by materials application, such as the generation of tertiary dentin or antibacterial effects. Finally, tissue regeneration may be supported and promoted by the use of various suitable materials (matrices/scaffolds) into which stem cells can migrate or be seeded, leading to cell differentiation and the generation of new tissue. These new dental materials must also fulfill additional requirements such as controlled degradability in order to be suitable for clinical use. Clearly, the field of material tissue interaction is complex and comprises a wide range of issues. To be successful as dentists in the future, practitioners should remain informed of these important new developments and have the argumentative competence to both properly advise and treat their patients.

Establishment of an optimized ex vivo system for artificial root canal infection evaluated by use of sodium hypochlorite and the photodynamic therapy.

AIM: To establish a refined model of artificially infected root canals and confirm its suitability as a sensitive ex vivo method to assess the efficacy of disinfecting agents. Disinfection was evaluated using sodium hypochlorite (NaOCl), either blocked or unblocked by sodium thiosulphate, and a recently promoted method of disinfection, the antibacterial photodynamic therapy (PDT). METHODOLOGY: The roots of bovine incisors were sectioned into three parts, the canals of coronal and middle regions were filled with a suspension of Enterococcus faecalis and the apical region with culture medium. After 7 days, coronal sections were disinfected using NaOCl (0.5%, 1.0% and 3.0% for 30, 60 and 600 s) or a system for photoactivated chemotherapy (PACT; Cumdente, Tübingen, Germany) for antibacterial PDT. Apical sections served as sterile controls and middle sections as bacterial growth controls. In half of the NaOCl-treated specimens, disinfection was arrested. Dentine chips from biopsies at different depths from the central canal towards the periphery were plated and assessed for colony-forming units (CFU). Disinfection was considered biologically relevant if the reduction of CFU was at least three log10 orders of magnitude. RESULTS: Colony-forming units of 10³ - 104 in growth controls indicated effective artificial infection. A biologically relevant reduction of CFU was accomplished with unblocked NaOCl, but not after blocking with NaOCl nor with PDT. CONCLUSIONS: The system reliably detected disinfection of the root canal and dentinal tubules and proved suitable for ex vivo testing of root canal disinfection. The effect of NaOCl depended on the duration of impact. Under the present experimental conditions, the antibacterial PDT system did not achieve sufficient disinfection.

TWIST1 promotes the odontoblast-like differentiation of dental stem cells.

Stem cells derived from the dental pulp of extracted human third molars (DPSCs) have the potential to differentiate into odontoblasts, osteoblasts, adipocytes, and neural cells when provided with the appropriate conditions. To advance the use of DPSCs for dentin regeneration, it is important to replicate the permissive signals that drive terminal events in odontoblast differentiation during tooth development. Such a strategy is likely to restore a dentin matrix that more resembles the tubular nature of primary dentin. Due to the limitations of culture conditions, the use of ex vivo gene therapy to drive the terminal differentiation of mineralizing cells holds considerable promise. In these studies, we asked whether the forced expression of TWIST1 in DPSCs could alter the potential of these cells to differentiate into odontoblast-like cells. Since the partnership between Runx2 and Twist1 proteins is known to control the onset of osteoblast terminal differentiation, we hypothesized that these genes act to control lineage determination of DPSCs. For the first time, our results showed that Twist1 overexpression in DPSCs enhanced the expression of DSPP, a gene that marks odontoblast terminal differentiation. Furthermore, co-transfection assays showed that Twist1 stimulates Dspp promoter activity by antagonizing Runx2 function in 293FT cells. Analysis of our in vitro data, taken together, suggests that lineage specification of DPSCs can be modulated through ex vivo gene modifications.

Scaffolds for dental pulp tissue engineering.

For tissue engineering strategies, the choice of an appropriate scaffold is the first and certainly a crucial step. A vast variety of biomaterials is available: natural or synthetic polymers, extracellular matrix, self-assembling systems, hydrogels, or bioceramics. Each material offers a unique chemistry, composition and structure, degradation profile, and possibility for modification. The role of the scaffold has changed from passive carrier toward a bioactive matrix, which can induce a desired cellular behavior. Tailor-made materials for specific applications can be created. Recent approaches to generate dental pulp rely on established materials, such as collagen, polyester, chitosan, or hydroxyapatite. Results after transplantation show soft connective tissue formation and newly generated dentin. For dentin-pulp-complex engineering, aspects including vascularization, cell-matrix interactions, growth-factor incorporation, matrix degradation, mineralization, and contamination control should be considered. Self-assembling peptide hydrogels are an example of a smart material that can be modified to create customized matrices. Rational design of the peptide sequence allows for control of material stiffness, induction of mineral nucleation, or introduction of antibacterial activity. Cellular responses can be evoked by the incorporation of cell adhesion motifs, enzyme-cleavable sites, and suitable growth factors. The combination of inductive scaffold materials with stem cells might optimize the approaches for dentin-pulp complex regeneration.