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This study aimed to design sustained released biodegradable calcium carbonate nanoparticles loaded with chlorhexidine (CHX-loaded NPs) and to investigate the early osteogenic differentiation and antimicrobial effects on the important bacteria involved in infections of dental implants. The microemulsion method was used to prepare the calcium carbonate nanoparticles loaded with chlorhexidine. The prepared nanoparticles were characterized using conventional methods. The release pattern determination and the biodegradation test were performed for the prepared nanoparticles. For the early osteogenic differentiation test of the prepared nanoparticles, alkaline phosphatase (ALP) activity was detected in human dental pulp stem cells (HDPSCs). The antimicrobial effects of the nanoparticles were evaluated against Escherichia coli, Streptococcus mutans, Enterococcus faecalis, Staphylococcus aureus, and Pseudomonas aeruginosa. The sizes of free calcium carbonate nanoparticles and CHX-loaded NPs were 105 ± 1.63 and 118 ± 1.47 nm and their zeta potentials were - 27 and - 36, respectively. A 50% degradation of nanoparticles was achieved after 100 days. These nanoparticles showed a two-stage sustained release pattern in vitro. Microscopic images revealed that the morphology of free calcium carbonate nanoparticles primarily took on a spherical calcite form, while CHX-loaded NPs predominantly exhibited a cauliflower-like vaterite polymorph. The nanoparticles increased the activity of ALP in cells in two weeks significantly (p < 0.05). Antimicrobial and antibiofilm results showed an efficient effect of the prepared nanoparticle against the studied bacteria. Calcium carbonate nanoparticles are an efficient multifunctional vector for chlorhexidine and can be used as a bioactive antibacterial agent against various oral microorganisms to prevent implant infections., (© 2024. The Author(s).)

Objective: Most endodontic diseases are bacterium-mediated inflammatory or necrotic process induced by contaminated dental pulp. Although great advances are being performed to obtain more efficient antibacterial strategies for persistent infections, most studies lack of representative models to test their antibacterial effects and their outcomes cannot be promptly translated to clinical practice. Therefore, this study aimed to refine an ex vivo endodontic biofilm model combining human tooth, computer guided design and 3D printing to obtain a more reproducible and predictable model., Methods: Monoradicular teeth were cut using three different methods: hand-held (HCC), mechanical precision (MPC) and computer aid guided cutting (CGC). Then, blocks were reassembled. The different model preparations were assessed in terms of dimensional tolerance, surface analysis, liquid tightness and Enterococcus faecalis biofilm development for 21 days, which was studied by metabolic assays and confocal microscopy. Then, the proposed model was validated using different commercial disinfecting treatments., Results: CGC exhibited significantly lower deviation and surface without defects compared to HHC and MPC, leading to superior liquid tightness. Similarly, mature biofilms with high metabolic activity and vitality were observed in all conditions, CGC showing the lowest variation. Regarding the model validation, all antibacterial treatments resulted in the complete eradication of bacteria in the standard 2D model, whereas commercial treatments exhibited varying levels of efficacy in the proposed ex vivo model, from moderately reduction of metabolic activity to complete elimination of biofilm., Conclusions: The novel guided approach represents a more reliable, standardized, and reproducible model for the evaluation of endodontic disinfecting therapies., Clinical Significance: During antibacterial treatment development, challenging 3D models using teeth substrates to test antibacterial treatments novel guided approach represents a more reliable, standardized, and reproducible model for the evaluation of endodontic disinfecting therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: Microbial pathogens invade various human organs, including the oral cavity. Candida albicans (C.a) and Streptococcus mutans (S.m) served respectively as representative oral pathogenic fungi and bacteria to stimulate dental pulp stem cells (DPSCs) and to screen the DPSC subcluster that specifically responded to fungal infection., Methods: DPSCs were obtained from the impacted third molars of six healthy subjects. Then, cells were mixed and divided into three samples, two of which were stimulated with C.a and S.m, respectively; the third sample was exposed to cell medium only (Ctrl). Single-cell mRNA sequencing analysis of treated DPSCs was performed., Results: DPSCs were composed of four major clusters of which one, DPSC.7, exhibited unique changes compared to those of other subclusters. The DPSC.7 cell percentage of the C.a sample was twice those of the Ctrl and S.m samples. DPSC.7 cells expressed genes associated with the response to reactive oxygen species (ROS) response. DPSC.7 subgroup cells established characteristic aggregation under the stimulation of different pathogens in UMAP. The MAPK/ERK1/2 and NF-κB pathways were up-regulated, DUSP1/5/6 expressions were suppressed, FOS synthesis was activated, the immune-related pathway was induced, and the levels of cytokines, including IL-6 and CCL2 , were up-regulated in DPSC.7 cells when stimulated with C.a., Conclusions: Our study analyzed the cellular and molecular properties of DPSCs infected by oral fungi and bacteria with single-cell RNA sequencing. A subcluster of DPSCs responded specifically to infections with different pathogens, activating the MAPK and NF-κB pathways to induce immune responses via the ROS pathway. This suggests novel treatment strategies for fungal infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Xu, Liu, Zhang, Li, Zhang, Li, Zhang, Yue, Li, Lin, Zou and Chen.)

Background: Pulpitis primarily arises from the pulp space infection by oral microbiota. Vital pulp therapy is a minimally invasive approach that relies on assessing the severity of pulpal inflammation to facilitate repair. However, the current evaluation methods prescribed by the American Association of Endodontics are subjective, leading to ambiguity in assessment. Therefore, this review aims to explore molecular strategies for evaluating the severity of pulpal inflammation to accurately predict the success of pulp vitality preservation in clinical settings., Methodology: This review was conducted by searching relevant keywords, such as irreversible pulpitis, pulpitis biomarkers, molecular diagnosis, inflammation, and genomic strategies, in databases such as PubMed, Web of Science, and Scopus to address the subjective nature of diagnosis. The data included in this review were collected up to April 2023. The literature search revealed well-documented limitations in clinically assessing the pulp inflammatory. Molecular approaches that aid in clinical differentiation between irreversible and reversible pulpitis may potentially enhance favorable outcomes in vital pulp therapy. Non-invasive diagnostic methods for pulpal assessment would also be valuable for determining whether the inflamed pulp is reversible, irreversible, or necrotic., Conclusion: The present review examines the various molecular diagnostic approaches that have revolutionized the medical field and are considered the most promising empirical methodologies for the proactive detection of pulpal diseases. It also provides comprehensive insights into the current diagnostic methods, associated challenges, next-generation strategies, and future directions for diagnosing the severity of pulp inflammation., Competing Interests: Declaration of competing interest Revolutionizing the diagnosis of Irreversible Pulpitis – Current Strategies and Future Directions, All authors are required to disclose any COI within the period of 12 months prior to the submission of any manuscript in the subject matter of which any company, entity, or organization has an interest. - Nil, The corresponding author is required to complete this form with information from all the authors listed in the manuscript., (Copyright © 2024 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

Background: Dental pulp in a confined environment, with little connection to the outside and only a small distribution of immune cells, provides a good research model for investigating how cells respond to bacterial infections through cytokines., Methods: The data of single-cell transcriptome sequencing of healthy and inflamed pulp tissue were downloaded from the GEO dataset. The expression character of 79 cytokines was analyzed based on the expression matrix., Results: The cytokine secretion profiles of the two populations of pulp cells in healthy dental pulp were associated with vascularization and nervous system development, as well as immune cell regulation. For the three populations of pulp stem cells with stem cell activity in the dental pulp, the secretion of cytokines related to nervous system development, regulation of endothelial cell proliferation and migration, and regulation of immune cell function comprised the characteristics that we observed. The cytokines secreted by T cells and macrophages were more of an immune reserve against pathogenic microorganisms. In the inflammatory state, the spectrum of cytokines secreted by various types of cells in the dental pulp tended to be identical, such that it mainly resisted pathogenic microorganisms., Conclusions: The cytokine secretion profiles of various cell types in healthy and inflamed dental pulp at the single-cell level are summarized., (© 2024 John Wiley & Sons Ltd.)

Introduction: Dental caries results from an ecologic shift within the dental biofilm from a balanced population of microorganisms to an acidogenic, aciduric, and cariogenic microbiological population developed and maintained by frequent consumption of fermentable dietary carbohydrates. Total caries removal (TCR) of deep lesion may result in pulpal exposure requiring more invasive treatment. Hence, current pediatric dentistry has shifted to minimally invasive treatment that avoids more complex, time-consuming procedure, and the child's discomfort., Aim: The aim of this study is to evaluate and compare clinical performance and radiographic changes after complete and incomplete caries removal procedures., Materials and Methods: The study was conducted on 60 primary molars in children aged 6-9 years. Selected 60 primary molars were randomly divided into two groups. Group 1 (PCR): infected dentin was removed, while the affected dentin was maintained on the pulpal wall. Group 2 (TCR): both infected and affected dentin were removed through low-speed carbide bur and hand excavator. Teeth were evaluated at 4 and 6 months clinically and radiographically., Results: The proportion was compared using Fisher's exact test. The Statistical Package for the Social Sciences version 21 was used for analysis. The level of significance was kept at 5%., Conclusion: The clinical and radiographic success rates of ICR and CCR in primary teeth with deep carious lesions were high and did not differ significantly, indicating that the retention of carious dentin does not interfere with pulp vitality. Thus, ICR is a reliable minimally invasive approach that might replace the CCR in primary teeth when correctly indicated., (Copyright © 2024 Copyright: © 2024 Journal of Indian Society of Pedodontics and Preventive Dentistry.)

Introduction: During pulpitis, as bacteria penetrate deeper into the dentin and pulp tissue, a pulpal innate immune response is initiated. However, the kinetics of the immune response, how this relates to bacterial infiltration during pulpitis and an understanding of the types of immune cells in the pulp is limited., Methods: Dental pulp exposure in the molars of mice was used as an animal model of pulpitis. To investigate the kinetics of immune response, pulp tissue was collected from permanent molars at different time points after injury (baseline, day 1, and day 7). Flow cytometry analysis of CD45+ leukocytes, including macrophages, neutrophils monocytes, and T cells, was performed. 16S in situ hybridization captured bacterial invasion of the pulp, and immunohistochemistry for F4/80 investigated spatial and morphological changes of macrophages during pulpitis. Data were analyzed using two-way ANOVA with Tukey's multiple comparisons., Results: Bacteria mostly remained close to the injury site, with some expansion towards noninjured pulp horns. We found that F4/80 + macrophages were the primary immune cell population in the healthy pulp. Upon injury, CD11b  +  Ly6G high neutrophils and CD11b  +  Ly6G int Ly6C int monocytes constituted 70-90% of all immune populations up to 7 days after injury. Even though there was a slight increase in T cells at day 7, myeloid cells remained the main drivers of the immune response during the seven-day time period., Conclusions: As bacteria proliferate within the pulp chamber, innate immune cells, including macrophages, neutrophils, and monocytes, predominate as the major immune populations, with some signs of transitioning to an adaptive immune response., (Copyright © 2023. Published by Elsevier Inc.)

Background/purpose: The signaling mechanisms for Porphyromonas gingivalis lipopolysaccharide (PgLPS)-induced inflammation in human dental pulp cells are not fully clarified. This in vitro study aimed to evaluate the involvement of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in PgLPS-induced pulpal inflammation., Methods: Human dental pulp cells (HDPCs) were challenged with PgLPS with or without pretreatment and coincubation with a PI3K/Akt inhibitor (LY294002). The gene or protein levels of PI3K, Akt, interleukin (IL)-6, IL-8, alkaline phosphatase (ALP), osteocalcin and osteonectin were analyzed by reverse transcription polymerase chain reaction (PCR), real-time PCR, western blotting, and immunofluorescent staining. In addition, an enzyme-linked immunosorbent assay was used to analyze IL-6 and IL-8 levels in culture medium., Results: In response to 5 μg/ml PgLPS, IL-6, IL-8, and PI3K, but not Akt mRNA expression of HDPCs, was upregulated. IL-6, IL-8, PI3K, and p-Akt protein levels were stimulated by 10-50 μg/ml of PgLPS in HDPCs. PgLPS also induced IL-6 and IL-8 secretion at concentrations higher than 5 μg/ml. Pretreatment and co-incubation by LY294002 attenuated PgLPS-induced IL-6 and IL-8 mRNA expression in HDPCs. The mRNA expression of ALP, but not osteocalcin and osteonectin, was inhibited by higher concentrations of PgLPS in HDPCs., Conclusion: P. gingivalis contributes to pulpal inflammation in HDPCs by dysregulating PI3K/Akt signaling pathway to stimulate IL-6 and IL-8 mRNA/protein expression and secretion. These results are useful for understanding the pulpal inflammation and possible biomarkers of inflamed pulp diagnosis and treatment., Competing Interests: Declaration of competing interest The authors have no conflicts of interest relevant to this article., (Copyright © 2022 Formosan Medical Association. Published by Elsevier B.V. All rights reserved.)

The prevalence of dental caries in the Mexican adult population aged 20 to 85 years is around 93.3%, and 50% in Mexican children and adolescents. Worldwide, it is the most common non-communicable disease. One of the main etiological factors for dental caries is the oral microbiome and changes in its structure and function, with an expansion of pathogenic bacteria like Streptococcus mutans . The exposed dental pulp tissue triggers an innate immune response to counteract this bacterial invasion. The relation between oral dysbiosis and innate immune responses remains unclear. We aimed to understand the relationship between innate immune response and the oral microbiota by quantifying the expression of Toll-like receptors (TLRs) and proinflammatory markers (cytokines and a chemokine) in dental pulp tissue, either exposed or not to carious dentin, and to correlate this information with the oral microbiome found in healthy teeth and those with moderate caries. RNA was purified from pulp tissue, subjected to RT-qPCR and analysed with the ΔΔCt method. Supragingival dental plaque of non-carious teeth and dentin of carious teeth were subjected to 16S targeted sequencing. Principal coordinate analysis, permutational multivariate ANOVA, and linear discriminant analysis were used to assess differences between non-carious and carious teeth. Correlations were assessed with Spearman´s test and corrected for multiple comparisons using the FDR method. The relative abundance (RA) of Lactobacillus, Actinomyces, Prevotella , and Mitsuokella was increased in carious teeth; while the RA of Haemophilus and Porphyromonas decreased. Olsenella and Parascardovia were only detected in carious teeth. Significant overexpression of interleukin 1 beta (IL1 β), IL6, and CXCL8 was detected in pulp tissue exposed to carious dentin. IL1β correlated positively with TLR2 and Actinomyces ; yet negatively with Porphyromonas. These findings suggest that immune response of pulp tissue chronically exposed to cariogenic microbiome is triggered by proinflammatory cytokines IL1β and IL6 and the chemokine CXCL8., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gómez-García, López-Vidal, Pinto-Cardoso and Aguirre-García.)

Root canal therapy has been practiced ever since 1928 and the success rate has tremendously increased over the years owing to various advancements in the field. One main reason is the complete understanding of the microbiology involved in the endodontic pathology. This has helped us to modify the conventional treatment plans and effectively combat the microorganisms. Now, studies are aiming to explore the characteristics of the "most" resistant organism and the methods to eliminate them. This article gives an insight of the microbiology involved in endodontic pathology and discusses its role in our treatment procedure. Information from original reviews listed in PubMed, published from 1995 to 2010, has been mainly included in this review. [ABSTRACT FROM AUTHOR]

Background: Citation analysis has emerged to play a significant role in recognition of the most useful areas of research. Endodontic microbiology has been a topic of interest for endodontists as well as periodontists and oral surgeons. This bibliometric analysis is aimed at identifying and reporting the characteristics of the top 50 cited articles on endodontic microbiology., Methods: The articles were identified through a search on Web of Science (WoS), property of Clarivate Analytics database published on endodontic microbiology. The citation information of the selected articles was recorded. The Journal of Endodontics , International Endodontic Journal , Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontology , Dental Traumatology , and Australian Endodontic Journal were searched in the search title. Descriptive and bivariate analyses were performed using a statistical software package SPSS. Statistical analysis was performed using Shapiro-Wilk, Kruskal-Wallis, Post hoc, Mann-Kendall trend, and Spearman-rank tests., Results: The 50 most cited articles were published from 1965 to 2012 with citation count varying from 1065 to 103 times. The total citation counts of articles recorded were 11,525 (WoS), 12,602 (Elseviers' Scopus), and 28,871 (Google Scholar). The most prolific years in terms of publications were 2001, 2002, and 2003, with five publications each, followed by 2005 with four. The year with most citations was 1998, with 1,330 citations, followed by 1965 and 2001, with 1,065 and 1,015 citations, respectively. A total of 136 authors contributed to the top 50 most cited articles with 27 corresponding institutions from 12 different countries. The most common methodological design was in vitro study, followed by clinic-laboratory study, literature review, systematic review and meta-analysis, and animal study., Conclusions: The present study provided a detailed list of the top 50 most cited and classic articles on microbiology in endodontics. This will help researchers, students, and clinicians in the field of endodontics as an impressive source of information., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Mohmed Isaqali Karobari et al.)

Bacterial/fungal biofilm-mediated persistent endodontic infections (PEIs) are one of the most frequent clinical lesions in the oral cavity, resulting in apical periodontitis and tooth damage caused by loss of minerals. The conventional root canal disinfectants are poorly bio-safe and harmful to teeth and tissues, making them ineffective in treating PEIs. The development of nanomaterials is emerging as a promising strategy to eradicate disease-related bacteria/fungi. Herein, glucose oxidase (GOx)-modified magnetic nanoparticles (MNPs) were synthesized via a facile and versatile route for investigating their effects on removing PEI-related bacterial/fungal biofilms. It is found that GOx was successfully immobilized on the MNPs by detecting the changes in the diameter, chemical functional group, charge, and magnetic response. Further, we demonstrate that GOx-modified MNPs (GMNPs) exhibit highly effective antibacterial activity against Enterococcus faecalis and Candida albicans . Moreover, the antibacterial/fungal activity of GMNPs is greatly dependent on their concentrations. Importantly, when placed in contact with bacterial/fungal biofilms, the dense biofilm matrix is destructed due to the movement of GMNPs induced by the magnetic field, the formation of reactive oxygen species, and nutrient starvation induced by GOx. Also, the in vitro experiment shows that the as-prepared GMNPs have excellent cytocompatibility and blood compatibility. Thus, GMNPs offer a novel strategy to treat bacteria/fungi-associated PEIs for potential clinical applications.

The macroscopic and microscopic anatomy of the oral cavity is complex and unique in the human body. Soft-tissue structures are in close interaction with mineralized bone, but also dentine, cementum and enamel of our teeth. These are exposed to intense mechanical and chemical stress as well as to dense microbiologic colonization. Teeth are susceptible to damage, most commonly to caries, where microorganisms from the oral cavity degrade the mineralized tissues of enamel and dentine and invade the soft connective tissue at the core, the dental pulp. However, the pulp is well-equipped to sense and fend off bacteria and their products and mounts various and intricate defense mechanisms. The front rank is formed by a layer of odontoblasts, which line the pulp chamber towards the dentine. These highly specialized cells not only form mineralized tissue but exert important functions as barrier cells. They recognize pathogens early in the process, secrete antibacterial compounds and neutralize bacterial toxins, initiate the immune response and alert other key players of the host defense. As bacteria get closer to the pulp, additional cell types of the pulp, including fibroblasts, stem and immune cells, but also vascular and neuronal networks, contribute with a variety of distinct defense mechanisms, and inflammatory response mechanisms are critical for tissue homeostasis. Still, without therapeutic intervention, a deep carious lesion may lead to tissue necrosis, which allows bacteria to populate the root canal system and invade the periradicular bone via the apical foramen at the root tip. The periodontal tissues and alveolar bone react to the insult with an inflammatory response, most commonly by the formation of an apical granuloma. Healing can occur after pathogen removal, which is achieved by disinfection and obturation of the pulp space by root canal treatment. This review highlights the various mechanisms of pathogen recognition and defense of dental pulp cells and periradicular tissues, explains the different cell types involved in the immune response and discusses the mechanisms of healing and repair, pointing out the close links between inflammation and regeneration as well as between inflammation and potential malignant transformation.

Objectives: Depicting past epidemics currently relies on DNA-based detection of pathogens, an approach limited to pathogens with well-preserved DNA sequences. We used paleoserology as a complementary approach detecting specific antibodies under a mini line-blot format including positive and negative control antigens., Methods: Mini line blot assay incorporated skim milk as negative control, Staphylococcus aureus as positive control, and antigens prepared from lice-borne pathogens Rickettsia prowazekii, Borrelia recurrentis, Bartonella quintana, and Yersinia pestis. Paleoserums were extracted from rehydrated dental pulp recovered from buried individuals. Mini line blots observed with the naked eye, were quantified using a scanner and appropriate software. Paleoserology was applied to the indirect detection of lice-borne pathogens in seven skeletons exhumed from a 16th-17th century suspected military burial site (Auxi-le-Château); and 14 civils exhumed from a 5th-13th century burial site (Saint-Mont). Direct detection of pathogens was performed using quantitative real-time PCR., Results: In Auxi-le-Château, paleoserology yielded 7/7 interpretable paleoserums including 7/7 positives for B. recurrentis including one also positive for B. quintana. In Saint-Mont, paleoserology yielded 8/14 interpretable paleoserums and none reacted against any of the four pathogens. Antibodies against R. prowazekii and Y. pestis were not detected. The seroprevalence was significantly higher in the military burial site of Auxi-le-Château than in the civil burial site of Saint-Mont. Real-time PCR detection of B. quintana yielded 5/21 positive (3 at Saint-Mont and 2 at Auxi-le-Château) whereas B. recurrentis was not detected., Conclusions: Paleoserology unmasked an outbreak of relapsing B. recurrentis fever in one 16th - 17th century military garrison, missed by real-time PCR. Paleoserology offers a new tool for investigating past epidemics, in complement to DNA sequence-based approaches., (© 2020 Wiley Periodicals LLC.)

During the two World Wars, Bartonella quintana was responsible for trench fever and is now recognised as an agent of re-emerging infection. Many reports have indicated widespread B. quintana exposure since the 1990s. In order to evaluate its prevalence in ancient populations, we used real-time PCR to detect B. quintana DNA in 400 teeth collected from 145 individuals dating from the 1st to 19th centuries in nine archaeological sites, with the presence of negative controls. Fisher's exact test was used to compare the prevalence of B. quintana in civil and military populations. B. quintana DNA was confirmed in a total of 28/145 (19.3%) individuals, comprising 78 citizens and 67 soldiers, 20.1% and 17.9% of which were positive for B. quintana bacteraemia, respectively. This study analysed previous studies on these ancient samples and showed that the presence of B. quintana infection followed the course of time in human history; a total of 14/15 sites from five European countries had a positive prevalence. The positive rate in soldiers was higher than those of civilians, with 20% and 18.8%, respectively, in the 18th and 19th centuries, but the difference in frequency was not significant. These results confirmed the role of dental pulp in diagnosing B. quintana bacteraemia in ancient populations and showed the incidence of B. quintana in both civilians and soldiers., Competing Interests: The authors have declared that no competing interests exist.

Hydrogen peroxide photolysis-based antimicrobial chemotherapy that utilizes ultraviolet-A irradiation (UVA-H 2 O 2 photolysis) has been previously proposed as a method of treatment of cariogenic biofilm. Therefore, in the present study, we aimed to assess time-dependent reactions in the dental pulp of rats after UVA-H 2 O 2 photolysis. Maxillary first molars were treated. UVA irradiation (wavelength: 365 nm) with 3 wt% H 2 O 2 was performed for 90 s at a radiant emittance of 500-2000 mW/cm 2 on the rats for 3 consecutive days or only 1 day. The animals were sacrificed at Days 1, 3, 7, and 21 after the treatment for the histological evaluation of inflammatory cells and immunohistochemistry of heat shock protein (HSP)-25, a marker of odontoblasts. Tertiary dentin formation was evaluated at Day 21 by histomorphometry and micro-CT analysis. UVA-H 2 O 2 photolysis elicited little infiltration of inflammatory cells, but disturbances in the odontoblast layer and/or presence of localized degenerative tissue were observed on Day 3. This condition was followed by a healing process that was characterized by the reappearance of HSP-25 positive odontoblast-like cells at Day 7 and tertiary dentin formation at Day 21. The amount of tertiary dentin formed was dependent on the intensity of treatment; repeated UVA irradiations of H 2 O 2 at 2000 mW/cm 2 resulted in the largest amount of tertiary dentin formation at the pulp horn regions. Our findings suggest that UVA-H 2 O 2 photolysis treatment can be used to treat dental caries clinically because the post-treatment inflammatory reaction was minimal and tertiary dentin formation was substantial, which may prove effective in protecting dental pulp from external irritants. As a cautionary consideration, the radiant emittance of the UVA irradiation should be carefully optimized before clinical application., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Groundbreaking studies conducted in the mid-1980s demonstrated the possibility of sequencing ancient DNA (aDNA), which has allowed us to answer fundamental questions about the human past. Microbiologists were thus given a powerful tool to glimpse directly into inscrutable bacterial history, hitherto inaccessible due to a poor fossil record. Initially plagued by concerns regarding contamination, the field has grown alongside technical progress, with the advent of high-throughput sequencing being a breakthrough in sequence output and authentication. Albeit burdened with challenges unique to the analysis of bacteria, a growing number of viable sources for aDNA has opened multiple avenues of microbial research. Ancient pathogens have been extracted from bones, dental pulp, mummies and historical medical specimens and have answered focal historical questions such as identifying the aetiological agent of the black death as Yersinia pestis . Furthermore, ancient human microbiomes from fossilized faeces, mummies and dental plaque have shown shifts in human commensals through the Neolithic demographic transition and industrial revolution, whereas environmental isolates stemming from permafrost samples have revealed signs of ancient antimicrobial resistance. Culminating in an ever-growing repertoire of ancient genomes, the quickly expanding body of bacterial aDNA studies has also enabled comparisons of ancient genomes to their extant counterparts, illuminating the evolutionary history of bacteria. In this review we summarize the present avenues of research and contextualize them in the past of the field whilst also pointing towards questions still to be answered.

Introduction: Dental pulp with special structure has become a good reference sample in paleomicrobiology-related blood-borne diseases, many pathogens were detected by different methods based on the diagnosis of nucleic acids and proteins., Objectives: This review aims to propose the preparation process from ancient teeth collection to organic molecule extraction of dental pulp and summary, analyze the methods that have been applied to detect septicemic pathogens through ancient dental pulps during the past 20 years following the first detection of an ancient microbe., Methods: The papers used in this review with two main objectives were obtained from PubMed and Google scholar with combining keywords: "ancient," "dental pulp," "teeth," "anatomy," "structure," "collection," "preservation," "selection," "photography," "radiography," "contamination," "decontamination," "DNA," "protein," "extraction," "bone," "paleomicrobiology," "bacteria," "virus," "pathogen," "molecular biology," "proteomics," "PCR," "MALDI-TOF," "LC/MS," "ELISA," "immunology," "immunochromatography," "genome," "microbiome," "metagenomics.", Results: The analysis of ancient dental pulp should have a careful preparation process with many different steps to give highly accurate results, each step complies with the rules in archaeology and paleomicrobiology. After the collection of organic molecules from dental pulp, they were investigated for pathogen identification based on the analysis of DNA and protein. Actually, DNA approach takes a principal role in diagnosis while the protein approach is more and more used. A total of seven techniques was used and ten bacteria (Yersinia pestis, Bartonella quintana, Salmonella enterica serovar Typhi, Salmonella enterica serovar Paratyphi C, Mycobacterium leprae, Mycobacterium tuberculosis, Rickettsia prowazeki, Staphylococcus aureus, Borrelia recurrentis, Bartonella henselae) and one virus (Anelloviridae) were identified. Y. pestis had the most published in quantity and all methods were investigated for this pathogen, S. aureus and B. recurrentis were identified by three different methods and others only by one. The combining methods interestingly increase the positive rate with ELISA, PCR and iPCR in Yersinia pestis diagnosis. Twenty-seven ancient genomes of Y. pestis and one ancient genome of B. recurrentis were reconstructed. Comparing to the ancient bone, ancient teeth showed more advantage in septicemic diagnosis. Beside pathogen identification, ancient pulp help to distinguish species., Conclusions: Dental pulp with specific tissue is a suitable sample for detection of the blood infection in the past through DNA and protein identification with the correct preparation process, furthermore, it helps to more understand the pathogens of historic diseases and epidemics., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Pulpitis is known as a typical inflammation of dental pulp tissue, and microorganisms of the oral microbiome are involved in this opportunistic infection. Studies indicated that several factors related to host response have a crucial role in pulpitis. Among these factors, inflammatory mediators of the immune system such as cytokines and chemokines contribute to pulpal defense mechanisms. A wide range of cytokines have been observed in dental pulp and these small molecules are able to trigger inflammation and participate in immune cell trafficking, cell proliferation, inflammation, and tissue damage in pulp space. Therefore, the aim of this review was to describe the role of cytokines in the pathogenesis of pulpitis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Helicobacter pylori (H. pylori) colonize the stomach and are considered an etiological agent of gastric cancer. The oral cavity is a transmission route to the stomach, but the exact site of colonization has not yet been explicated. Our study investigated the association between H. pylori infection and presence in oral samples., Methods: Dental pulp, supragingival plaque, and saliva from 192 patients visiting the Dentistry's outpatient clinic were collected for testing. The H. pylori ureA gene was identified via Nested PCR. Urine anti-H. pylori antibody test was utilized to detect infection., Results: Twenty-five subjects were found to be antibody-positive. PCR analysis of dental pulp revealed that 23 subjects possessed the ureA gene. Twenty-one subjects were positive for both antibodies and genes in dental pulp. PCR testing revealed that 2 subjects were positive in dental plaque but negative for saliva. The subjects positive for H. pylori in dental pulp expressed clinical signs of severe dental caries., Conclusions: H. pylori infected subjects expressed H. pylori in samples from the oral cavity. The main reservoir for infection within the oral cavity was determined to be dental pulp. Moreover, H. pylori are likely transmitted from dental caries to the root canal.

Dental caries is an infectious oral disease caused primarily by complex interactions of cariogenic oral flora (biofilm) with dietary carbohydrates on the tooth surface over time. Streptococcus mutans and Streptococcus sobrinus (S. mutans and S. sobrinus) are the most prevalent cariogenic species within the oral biofilm and considered the main etiological agents of caries. Pulp exposure and infection can be caused by trauma, carious lesion, and mechanical reasons. Pulp response to these exposures depends on the state of the pulp as well as the potential bacterial contamination of pulp tissue. Herein, we describe the process of using two in vivo rodent models to study the progression of dental caries and pulp disease: a nutritional microbial model and a pulp disease induction model. The progression of the carious lesion and pulpal infections in both models was assessed by micro-CT imaging and histomorphometric analysis. Moreover, the pulp disease induction models can be used to compare and assess the antibacterial and reparative properties of the different pulp capping materials.

Introduction: We evaluated the radiopacity and antibacterial properties of various concentrations of double antibiotic paste (DAP) containing barium sulfate (BaSO 4 ) or zirconium oxide (ZrO 2 ) radiopaque agents., Methods: The radiopacity of 1, 10, and 25 mg/mL DAP containing 30% (w/v) BaSO 4 or ZrO 2 , DAP-free radiopaque pastes, and commercially available radiopaque calcium hydroxide (Ca[OH] 2 ) were evaluated according to ISO 6876/2001 with slight modifications (n = 6 per group). Dentin samples (n = 70) infected anaerobically for 3 weeks with bacterial biofilms obtained from a root canal of an immature tooth with pulpal necrosis were treated with similar experimental pastes or received no treatment (n = 7). After 1 week, the pastes were rinsed off, and biofilm disruption assays were conducted. To show the residual antibacterial effects, sterile dentin samples (n = 70) were pretreated for 1 week with the same pastes (n = 7). The pastes were rinsed off, and the samples were immersed in phosphate-buffered saline for 24 hours and infected anaerobically with the same bacterial biofilm mentioned earlier for 3 weeks before conducting biofilm disruption assays. Sterile dentin blocks were used in both antibacterial analyses as negative control groups (n = 7). Wilcoxon rank sum tests were used for statistical analyses., Results: No tested concentrations of BaSO 4 DAP or ZrO 2 DAP showed significant differences from Ca(OH) 2 in radiopacity. However, all tested concentrations of BaSO 4 DAP, ZrO 2 DAP, and Ca(OH) 2 exhibited significant direct antibacterial effects. ZrO 2 DAP at 1 mg/mL and Ca(OH) 2 did not show significant residual antibacterial effects., Conclusions: BaSO 4 DAP at 1 mg/mL provided significantly superior residual antibacterial effects and comparable radiopacity with the commercially available Ca(OH) 2 ., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Aim: The aim of this in vivo study is to evaluate the antimicrobial efficacy of calcium hydroxide (Ca(OH) 2 ), chlorhexidine (CHX), and metronidazole gel as intracanal medicaments against aerobic and facultative anaerobic microorganisms found in root canals of human primary teeth with necrotic pulp., Setting and Design: It is a double-blinded randomized clinical trial., Materials and Methods: Pulp canals of 45 single-rooted primary maxillary anterior teeth with pulp necrosis in 34 children were included in the study. They were divided into three groups of 15 samples each: Group I: Ca(OH) 2 ; Group II: 1% CHX gel; and Group III: 1% metronidazole gel. Microbial count was obtained from each tooth at two different stages - (1) after instrumentation and (2) after placement of the medication. Statistical analysis using the SPSS 10.0 software program (SPSS Inc., Chicago, IL, USA) with Wilcoxon signed-rank test after grouping the samples was performed., Results: Ca(OH) 2 , 1% CHX gel, and 1% metronidazole gel were ineffective in completely eliminating aerobic and facultative anaerobic microorganism from root canal of human primary teeth with necrotic pulp., Conclusion: None of the commonly used intracanal medicaments, that is Ca(OH) 2 , 1% CHX gel, and 1% metronidazole gel, was effective in completely eliminating aerobic and facultative anaerobic microorganism from root canal of human primary teeth with necrotic pulp. Ineffectiveness of these medicaments against aerobic and facultative anaerobic microorganism has opened new door of research regarding the manner of bacterial growth in unfavorable environmental and nutritional conditions, the way root canal environment favors biofilm formation and the use of suitable intracanal medicaments against single and multispecies biofilms., Competing Interests: There are no conflicts of interest

Introduction: Biofilms are present in more than 70% of endodontically diseased teeth. Through the advancements in the next-generation sequencing (NGS) technologies, microbiome research has granted a deeper analysis of the microbial communities living in human hosts. Here, we reviewed previous studies that used NGS to profile the microbial communities of root canals., Methods: A total of 12 peer-reviewed articles from PubMed were identified and critically reviewed. The study criteria were as follows: NGS platforms, sequenced bacterial hypervariable regions, teeth diagnosis with available patient information, sample characteristics, collection method, and microbial signatures., Results: The most common NGS platforms used were 454 pyrosequencing (Roche Diagnostic Corporation, Risch-Rotkreuz, Switzerland) and Illumina-based technology (Illumina Inc, San Diego, CA). The hypervariable regions sequenced were between the V1 and V6 regions. The patient and sample population ranged from ages 12-76 years and asymptomatic and symptomatic teeth diagnosed with pulp necrosis with or without apical periodontitis. Microbial sampling was conducted directly from the infected pulp or the extracted teeth. The most abundant phyla were Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, and Fusobacteria. The most frequently detected genera were Prevotella, Fusobacterium, Porphyromonas, Parvimonas, and Streptococcus. Other notable microbial signatures at different taxa levels were identified but were widely variable between studies., Conclusions: Technologies based on high-throughput 16S ribosomal RNA NGS can aid in deciphering the complex bacterial communities of root canal biofilms. Thus far, only a few studies have been published with relatively small sample sizes, variable sample collection protocols, and community analyses methods. Future larger clinical studies are essential with validated standardized protocols for improved understanding of the pathogenic nature of bacterial biofilm communities in root canals., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Advances in culture-independent molecular biotechnologies have driven a greater appreciation for the function of mutualistic microorganisms in the maintenance of states of health in humans. The purpose of this study was to test the long-held hypothesis that healthy pulp lack bacteria., Methods: Strict inclusion criteria were used to identify 10 pristine teeth from 10 healthy patients that were scheduled to be electively extracted in compliance with an orthodontic treatment plan. Using a rigorous disinfection protocol to isolate the operating field, the pulp space was accessed, and pulp tissue was collected in vivo from each tooth using a barbed broach. Genomic DNA was extracted from each pulp sample and analyzed for the presence of bacterial DNA using universal 16S ribosomal RNA polymerase chain reaction primers and MiSeq sequencing (Illumina, San Diego, CA) of community amplicons., Results: One hundred percent (10/10) of the tested pulp tissues demonstrated the presence of bacterial DNA, with a mean of 343 operational taxonomic units per sample (range, 191-479). These were derived from 12 genera in which Ralstonia, Actinetobacter, and Staphylococcus were predominant (43%-78% of total community). None of the negative-field controls and none of the instruments used in the study tested positive for the presence of contaminating DNA., Conclusions: This study presents evidence to support the conclusion that the pulp spaces of pristine healthy teeth contain detectable bacterial DNA., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The origin of Yersinia pestis and the early stages of its evolution are fundamental subjects of investigation given its high virulence and mortality that resulted from past pandemics. Although the earliest evidence of Y. pestis infections in humans has been identified in Late Neolithic/Bronze Age Eurasia (LNBA 5000-3500y BP), these strains lack key genetic components required for flea adaptation, thus making their mode of transmission and disease presentation in humans unclear. Here, we reconstruct ancient Y. pestis genomes from individuals associated with the Late Bronze Age period (~3800 BP) in the Samara region of modern-day Russia. We show clear distinctions between our new strains and the LNBA lineage, and suggest that the full ability for flea-mediated transmission causing bubonic plague evolved more than 1000 years earlier than previously suggested. Finally, we propose that several Y. pestis lineages were established during the Bronze Age, some of which persist to the present day.

Introduction: The present study reported the histological events that occurred in the radicular pulp of human mature teeth in the presence of medium/deep untreated caries lesions, and those teeth with restorations or direct pulp capping, with particular emphasis on the morphology of the canal wall dentine and the odontoblast layer., Methods: Sixty-two teeth with medium/deep caries lesions, extensive restorations or after application of a direct pulp capping procedure were obtained from 57 subjects. Fourteen intact mature teeth served as controls. Stained serial sections were examined for the pulp conditions of the coronal pulp. The teeth were classified as those with pulpal inflammation, or those with healed pulps. Histological changes that occurred in the roots at the pulp-dentine junction were investigated in detail., Results: All teeth (100%) in the experimental group showed pathologic changes in the radicular pulp, with varying amounts of tertiary dentine on the canal walls and absence of odontoblasts. These changes were identified from different portions of the canal wall surface. Non-adherent calcifications in the pulp tissue were observed in more than half of the specimens. Changes that deviate from classically-perceived histological relationships of the pulp-dentine complex were also observed in the radicular pulps of 33.7% of the control teeth., Conclusion: When challenged by bacteria and bacterial by-products invading dentinal tubules, odontoblasts in the radicular pulp may undergo cell death, possibly by apoptosis. This phenomenon may be caused by progressive root-ward diffusion of bacterial by-products, cytokines or reactive oxygen species through the pulp connective tissue., Clinical Significance: Although the vitality of the dental pulp in teeth with deep dentinal caries may be maintained with direct pulp capping or pulpotomy, the repair tissue that is formed resembles mineralised fibrous connective tissues more than true tubular dentine., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Recently, dental pulp has been considered a possible source of infection of Helicobacter pylori (H. pylori) in children. We previously developed a novel PCR system for H. pylori detection with high specificity and sensitivity using primer sets constructed based on the complete genome information for 48 H. pylori strains. This PCR system showed high sensitivity with a detection limit of 1-10 cells when serial dilutions of H. pylori genomic DNA were used as templates. However, the detection limit was lower (10 2 -10 3 cells) when H. pylori bacterial DNA was detected from inflamed pulp specimens. Thus, we further refined the system using a nested PCR method, which was much more sensitive than the previous single PCR method. In addition, we examined the distribution and virulence of H. pylori in inflamed pulp tissue., Methods: Nested PCR system was constructed using primer sets designed from the complete genome information of 48 H. pylori strains. The detection limit of the nested PCR system was 1-10 cells using both H. pylori genomic DNA and bacterial DNA isolated from inflamed pulp specimens. Next, distribution of H. pylori was examined using 131 inflamed pulp specimens with the nested PCR system. In addition, association between the detection of H. pylori and clinical information regarding endodontic-infected teeth were investigated. Furthermore, adhesion property of H. pylori strains to human dental fibroblast cells was examined., Results: H. pylori was present in 38.9% of inflamed pulp specimens using the nested PCR system. H. pylori was shown to be predominantly detected in primary teeth rather than permanent teeth. In addition, samplings of the inflamed pulp were performed twice from the same teeth at 1- or 2-week intervals, which revealed that H. pylori was detected in most specimens in both samplings. Furthermore, H. pylori strains showed adhesion property to human dental fibroblast cells., Conclusion: Our results suggest that H. pylori colonizes inflamed pulp in approximately 40% of all cases through adhesion to human dental fibroblast cells.

This study evaluated antimicrobial photodynamic therapy (aPDT) as an adjunct to endodontic treatment. Ten uniradicular teeth (control group (CG) = 4 (2 and test group (TG) = 6) with primary endodontic infections, from both genders, between 17 and 65 years old, were analyzed. Microbiological samples were collected before and after chemical-mechanical instrumentation (CMI), after aPDT (for the TG), and after the removal of the temporary restorations (second session). In TG, the aPDT was performed with 100 μg mL -1 methylene blue and irradiated with low power laser (InGaAIP, 660 nm; 100 mW; 40 s) with a fiber-coupled optical laser. Another irradiation (3 J; 30 s; spot size of 3 mm 2 ) was performed in the gingiva close to the apical foramen. The PCR was performed, after previous whole-genome amplification, for Enterococcus faecalis, Candida genus and Bacteria domain. For TG, a positive tooth for Candida spp. before of the CMI presented negative results in subsequent samples. Additionally, E. faecalis species was present in four samples before CMI, two after CMI, in one after the aPDT and was not detected at the second session. aPDT may be an effective adjunct therapy, resulting in a reduction (P = 0.0286) of the incidence of E. faecalis before root canal obturation., (© 2017 The American Society of Photobiology.)

Regeneration of infected tissues is a globally challenging issue in medicine and dentistry. Common clinical therapies involving a complete removal of infected areas together with a treatment of antimicrobial drugs are often suboptimal. Biomaterials with anti-bacterial and pro-regenerative potential can offer a solution to this. Here we design a novel nanocarrier based on a mesoporous silicate-calcium glass by doping with Ag ions and simultaneously loading antimicrobial drugs onto mesopores. The nanocarriers could controllably release multiple ions (silver, calcium, and silicate) and drugs (tetracycline or chlorohexidine) to levels therapeutically relevant, and effectively internalize to human dental stem cells (∼90%) with excellent viability, ultimately stimulating odontogenic differentiation. The release of Ag ions had profound effects on most oral bacteria species through a membrane rupture, and the antibiotic delivery complemented the antibacterial functions by inhibiting protein synthesis. Of note, the nanocarriers easily anchored to bacteria membrane helping the delivery of molecules to an intra-bacterial space. When administered to an infected dentin-pulp defect in rats, the therapeutic nanocarriers effectively regenerated tissues following a complete bacterial killing. This novel concept of multiple-delivering ions and drug can be extensively applied to other infectious tissues that require relayed biological functions (anti-bacterial then pro-regenerative) for successful healing., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Dental pulp regeneration after pulp necrosis in immature teeth represents a major departure from traditional endodontic therapy of these conditions. Preliminary clinical attempts have shown the feasibility of developing mineralized repair tissue, which may provide a clinically acceptable outcome. However, this outcome may not provide sufficient host response and root strength to ensure the longevity of the involved teeth. It is not clear if these preliminary suboptimal results are caused by the inability to fully disinfect the pulp space or the absence of a suitable progenitor cell/scaffold template together with adequate vascularity. Moreover, it is not known to what degree the root canal system needs to be disinfected in order for clinical success to be evident. This article describes the current clinical strategies and protocols for the optimal disinfection and preparation of the pulp space environment to promote periapical healing as well as soft and hard tissue development after an infectious process. Current and future strategies for disinfecting the pulp space with minimal disruption of the necessary biological factors from dentin, the progenitor cells in periapical vital tissues, and the vascularity are discussed. The potential for success of pulp regeneration after necrosis and infection would transform the practice of endodontics, even for mature teeth. This is a goal worth pursuing because it would achieve the restoration of normal host responses in the pulp space and the regeneration of destroyed dental tissues., (Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Staphylococcus epidermidis is one of most prevalent in dental caries or dental pulp which has the capability of horizontal genetic transfer between different bacterial species in the oropharynx, suggesting that it may evolve with the dissemination of resistant determinants, This study was performed to molecularly characterize and differentiate S. epidermidis isolated from dental caries and healthy individual. Also, two important cytokines in inflammation were assayed caused due to S. epidermidis of health and dental caries sources. Dental caries strains were more resistant with high MIC 50 and MIC 90 value. These isolates also showed the presence of mecA gene and another virulence gene i. e sea and seb comparatively more than healthy individual isolates. SCCmec types, III and IV was more prevalent in dental caries isolates where an as healthy individual was more non-typable. Additionally, the quantity of IL-1β and IL-8 caused due to dental caries isolates was seen more which indicate dental caries isolates are able to induce. This study showed that S. epidermidis a normal flora of oropharyngeal are more diverse to those strains which cause dental caries. S. epidermidis owns a prodigious genetic plasticity that permits to obtain, lose or regulate genetic elements that provide compensations to improve its colonization in the host., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Chemical decomposition and fragmentation may limit the detection of ancient host and microbial DNA while some proteins can be detected for extended periods of time. We applied paleoproteomics on 300-year-old dental pulp specimens recovered from 16 individuals in two archeological funeral sites in France, comprising one documented plague site and one documented plague-negative site. The dental pulp paleoproteome of the 16 teeth comprised 439 peptides representative of 30 proteins of human origin and 211 peptides representative of 27 proteins of non-human origin. Human proteins consisted of conjunctive tissue and blood proteins including IgA immunoglobulins. Four peptides were indicative of three presumable Yersinia pestis proteins detected in 3/8 dental pulp specimens from the plague-positive site but not in the eight dental pulp specimens collected in the plague-negative site. Paleoproteomics applied to the dental pulp is a new and innovative approach to screen ancient individuals for the detection of blood-borne pathogens and host inflammatory response.

Antimicrobial peptides (AMPs) are short cationic host-defense molecules that provide the early stage of protection against invading microbes. They also have important modulatory roles and act as a bridge between innate and acquired immunity. The types and functions of oral AMPs were reviewed and experimental reports on the use of natural AMPs and their synthetic mimics in caries and pulpal infections were discussed. Natural AMPs in the oral cavity, predominantly defensins, cathelicidins and histatins, possess antimicrobial activities against oral pathogens and biofilms. Incomplete debridement of microorganisms in root canal space may precipitate an exacerbated immune response that results in periradicular bone resorption. Because of their immunomodulatory and wound healing potentials, AMPs stimulate pro-inflammatory cytokine production, recruit host defense cells and regulate immuno-inflammatory responses in the vicinity of the pulp and periapex. Recent rapid advances in the development of synthetic AMP mimics offer exciting opportunities for new therapeutic initiatives in root canal treatment and regenerative endodontics., Statement of Significance: Identification of new therapeutic strategies to combat antibiotic-resistant pathogens and biofilm-associated infections continues to be one of the major challenges in modern medicine. Despite the presence of commercialization hurdles and scientific challenges, interests in using antimicrobial peptides as therapeutic alternatives and adjuvants to combat pathogenic biofilms have never been foreshortened. Not only do these cationic peptides possess rapid killing ability, their multi-modal mechanisms of action render them advantageous in targeting different biofilm sub-populations. These factors, together with adjunctive bioactive functions such as immunomodulation and wound healing enhancement, render AMPs or their synthetic mimics exciting candidates to be considered as adjuncts in the treatment of caries, infected pulps and root canals., (Published by Elsevier Ltd.)

Introduction. Infection and apoptosis are combined triggers for inflammation in dental tissues. Octamer-binding transcription factor 4-B1 (OCT4B1), a novel spliced variant of OCT4 family, could respond to the cellular stress and possess antiapoptotic property. However, its specific role in dental pulpitis remains unknown. Methods. To investigate the effect of OCT4B1 on inflammation of dental pulp cells (DPCs), its expression in inflamed dental pulp tissues and DPCs was examined by in situ hybridization, real-time PCR, and FISH assay. OCT4B1 overexpressed DPCs model was established, confirmed by western blot and immunofluorescence staining, and then stimulated with Lipopolysaccharide (LPS). Apoptotic rate was determined by Hoechst/PI staining and FACS. Cell survival rate was calculated by CCK8 assay. Results. In situ hybridization, real-time PCR, and FISH assay revealed that OCT4B1 was extensively expressed in inflamed dental pulp tissues and DPCs with LPS stimulation. Western blot and immunofluorescence staining showed the expression of OCT4B1 and OCT4B increased after OCT4B1 transfection. Hoechst/PI staining and FACS demonstrated that less red/blue fluorescence was detected and apoptotic percentage decreased (3.45%) after transfection. CCK8 demonstrated that the survival rate of pCDH-OCT4B1-flag cells increased. Conclusions. OCT4B1 plays an essential role in inflammation and apoptosis of DPCs. OCT4B might operate synergistically with OCT4B1 to reduce apoptosis.

Descriptions of the pathologic changes in the pulp and associated apical structures of human immature teeth in response to deep caries are lacking in the literature., Objectives: This article describes the histologic events associated with the radicular pulp and the apical tissues of human immature teeth following pulp inflammation and necrosis., Methods: Twelve immature teeth with destructive caries lesions were obtained from 8 patients. Two intact immature teeth served as controls. Teeth were extracted for reasons not related to this study and immediately processed for histopathologic and histobacteriologic analyses. Serial sections were examined for the pulp conditions and classified as reversible or irreversible pulp inflammation, or pulp necrosis. Other histologic parameters were also evaluated., Results: In the 3 cases with reversible pulp inflammation, tissue in the pulp chamber showed mild to moderate inflammation and tertiary dentin formation related to tubules involved in the caries process. Overall, the radicular pulp tissue, apical papilla and Hertwig's epithelial root sheath (HERS) exhibited characteristics of normality. In the 3 cases with irreversible pulp inflammation, the pulps were exposed and severe inflammation occurred in the pulp chamber, with minor areas of necrosis and infection. Large areas of the canal walls were free from odontoblasts and lined by an atubular mineralized tissue. The apical papilla showed extremely reduced cellularity or lack of cells and HERS was discontinuous or absent. In the 6 cases with pulp necrosis, the coronal and radicular pulp tissue was necrotic and colonized by bacterial biofilms. The apical papilla could not be discerned, except for one case. HERS was absent in the necrotic cases., Conclusion: While immature teeth with reversible pulpitis showed histologic features almost similar to normal teeth in the canal and in the apical region, those with irreversible pulpitis and necrosis exhibited significant alterations not only in the radicular pulp but also in the apical tissues, including the apical papilla and HERS., Clinical Significance: Alterations in the radicular pulp and apical tissues help explain the outcome of current regenerative/reparative therapies and should be taken into account when devising more predictable therapeutic protocols for teeth with incomplete root formation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

It is not known to what extent residual infection may interfere with the success of pulp regeneration procedures. The aim of this study was to determine, radiographically and histologically, the effect of residual bacteria on the outcome of pulp regeneration mediated by a tissue-engineered construct as compared with traditional revascularization. Periapical lesions were induced in 24 canine teeth of 6 ferrets. After disinfection with 1.25% NaOCl and triple antibiotic paste, ferret dental pulp stem cells, encapsulated in a hydrogel scaffold, were injected into half the experimental teeth. The other half were treated with the traditional revascularization protocol with a blood clot scaffold. After 3 mo, block sections of the canine teeth were imaged radiographically and processed for histologic and histobacteriologic analyses. Associations between variables of interest were evaluated through mixed effects regression models. There were no significant differences between the 2 experimental groups in radiographic root development ( P > 0.05). There was a significant association between the presence of persistent periapical radiolucency and root wall thickness ( P = 0.02). There was also no significant difference in histologic findings between the 2 experimental groups ( P > 0.05). The presence of residual bacteria was significantly associated with lack of radiographic growth ( P < 0.001). The amount of dentin-associated mineralized tissue formed in teeth with residual bacteria was significantly less than in teeth with no residual bacteria ( P < 0.001). Residual bacteria have a critical negative effect on the outcome of regenerative endodontic procedures.

An ideal pulp-capping agent needs to have good biocompatibility and promote reparative dentinogenesis. Although the effects of capping agents on healthy pulp are known, limited data regarding their effects on bacterial contaminated pulp are available. This study aimed to evaluate the reaction of contaminated pulps to various capping agents to assist clinicians in making informed decisions. Human dental pulp (HDP) cell cultures were developed from extracted human molars. The cells were exposed to a bacterial cocktail comprising Porphyromonas gingivalis, Prevotella intermedia, and Streptococcus gordonii before being cocultured with capping agents such as mineral trioxide aggregate (MTA) Portland cement (PC), and Dycal. HDP cell proliferation was assayed by MTS colorimetric cell proliferation assay, and its differentiation was evaluated by real-time PCR for detecting alkaline phosphatase, dentin sialophosphoprotein, and osteocalcin expressions. MTA and PC had no apparent effect, whereas Dycal inhibited HDP cell proliferation. PC stimulated HDP cell differentiation, particularly when they were exposed to bacteria. MTA and Dycal inhibited differentiation, regardless of bacterial infection. In conclusion, PC was the most favorable agent, followed by MTA, and Dycal was the least favorable agent for supporting the functions of bacterial compromised pulp cells.

Cariopathogen Streptococcus mutans exists in infected dental pulp of deciduous teeth and is frequently linked with heart diseases. Organotypic (3D) dental pulp stem cell (DPSC) cultures/microtissues, developed to mimic the physiological conditions in vivo, were utilized to assess the bacterial impact on their (i) 3D structural configuration and (ii) recovery mechanisms. The cultures, developed in extracellular matrix (ECM) bio-scaffold (Matrigel™), interacted with WT and GFP-tagged bacterial biofilms by permitting their infiltration through the ECM. Challenged cell constructs were visualized by F-actin/nuclei staining. Their pluripotency (Sox2) and differentiation (osteocalcin) markers were assessed by immunocytochemistry. Secreted mineral was detected by alizarin red, and 3D structural arrangements were analysed by epi-fluorescence and confocal scanning microscopy. Bacterial biofilm/ECM-embedded DPSC interactions appeared in distinct areas of the microtissues. Bacterial attachment to the cell surface occurred without evidence of invasion. Surface architecture of the challenged versus unchallenged microtissues was apparently unaltered. However, significant increases in thickness (138.42 vs 106.51 µm) and bacterial penetration were detected in challenged structures causing canal-like microstructures with various diameters (12.94 -42.88 µm) and average diameter of 20.66 to 33.42 µm per microtissue. Challenged constructs expressed pluripotency and differentiation markers and secreted the mineral. Presented model shows strong potential for assessing pulp-pathogen interactions in vivo. S. mutans infiltrated and penetrated the microtissues but did not invade the cells or compromise major cell repair mechanisms. These findings would suggest reexamining the role of S. mutans as an endodontic pathogen and investigating DPSC resistance to its pathogenicity.

Introduction: Root canal disinfection and the establishment of an intracanal microenvironment conducive to the proliferation/differentiation of stem cells play a significant role in regenerative endodontics. This study was designed to (1) investigate the antimicrobial efficacy of triple antibiotic-containing nanofibers against a dual-species biofilm and (2) evaluate the ability of dental pulp stem cells (DPSCs) to adhere to and proliferate on dentin upon nanofiber exposure., Methods: Seven-day-old dual-species biofilm established on dentin specimens was exposed for 3 days to the following: saline (control), antibiotic-free nanofibers (control), and triple antibiotic-containing nanofibers or a saturated triple antibiotic paste (TAP) solution (50 mg/mL in phosphate buffer solution). Bacterial viability was assessed using the LIVE/DEAD assay (Molecular Probes, Inc, Eugene, OR) and confocal laser scanning microscopy. For cytocompatibility studies, dentin specimens after nanofiber or TAP (1 g/mL in phosphate buffer solution) exposure were evaluated for cell adhesion and spreading by actin-phalloidin staining. DPSC proliferation was assessed on days 1, 3, and 7. Statistics were performed, and significance was set at the 5% level., Results: Confocal laser scanning microscopy showed significant bacterial death upon antibiotic-containing nanofiber exposure, differing significantly (P < .05) from antibiotic-free fibers and the control (saline). DPSCs showed enhanced adhesion/spreading on dentin specimens treated with antibiotic-containing nanofibers when compared with its TAP counterparts. The DPSC proliferation rate was similar on days 1 and 3 in antibiotic-free nanofibers, triple antibiotic-containing nanofibers, and TAP-treated dentin. Proliferation was higher (9-fold) on dentin treated with antibiotic-containing nanofibers on day 7 compared with TAP., Conclusions: Triple antibiotic-containing polymer nanofibers led to significant bacterial death, whereas they did not affect DPSC attachment and proliferation on dentin., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Aim: This was to assess the antimicrobial effectiveness of pastes used as intracanal medicaments against Enterococcus faecalis in necrotic immature teeth., Method: An agar-disc diffusion method over a period of 30 days was used. The following medications were analysed: triple antibiotic paste (TAP) I; double antibiotic paste (DAP) I; DAP I with calcium hydroxide; calcium hydroxide paste with saline solution 0.9 %; calcium hydroxide paste with chlorhexidine 2 % gel; DAP II; DAP II with zinc oxide; DAP II with calcium hydroxide; and saline solution 0.9 % as control. The diameters of the halos of inhibition (in mm) of tested medicaments were determined and analysed with one-way analysis of variance and Tukey's post hoc tests., Results: TAP I (G1) was significantly more effective against E. faecalis, and showed the largest halo of inhibition during all the experiments. DAP groups (G2 and G6) also showed inhibiting bacterial growth with said inhibition remaining stable throughout the 30-day period. In contrast, G4 and G5 groups, in which Ca(OH)2 was added to saline and chlorhexidine, had no antibacterial effect., Conclusions: TAP and DAP showed better antibacterial efficacy and remained active for 30 days. Combination of Ca(OH)2 with antibiotics should be avoided, due to the possibility of antibiotic hydrolysis.

Streptococcus mutans is a major pathogen of dental caries. Collagen-binding proteins (CBPs) (approximately 120 kDa), termed Cnm and Cbm, are regarded as important cell surface antigens related to the adherence of S. mutans to collagenous tissue. Furthermore, CBP-positive S. mutans strains are associated with various systemic diseases involving bacteremia, such as infective endocarditis. Endodontic infection is considered to be an important cause of bacteremia, but little is known regarding the presence of S. mutans in dental pulp tissue. In the present study, the distribution and virulence of S. mutans in dental pulp tissues were investigated by focusing on CBPs. Adhesion and invasion properties of various S. mutans strains were analyzed using human dental pulp fibroblasts (HDPFs). CBP-positive strains had a significantly higher rate of adhesion to HDPFs compared with CBP-defective isogenic mutant strains (P<0.001). In addition, CBP-positive strains induced HDPF proliferation, which is a possible mechanism related to development of hyperplastic pulpitis. The distribution of S. mutans strains isolated from infected root canal specimens was then analyzed by PCR. We found that approximately 50% of the root canal specimens were positive for S. mutans. Approximately 20% of these strains were Cnm-positive, while no Cbm-positive strains were isolated. The Cnm-positive strains isolated from the specimens showed adhesion to HDPFs. Our results suggest that CBP-positive S. mutans strains exhibit high colonization in dental pulp. This could be a possible virulence factor for various systemic diseases.

Owing to the growing infectious diseases caused by eukaryotic and prokaryotic pathogens, it is urgent to develop novel antimicrobial agents against clinical pathogenic infections. Biofilm formation and invasion into the host cells are vital processes during pathogenic colonization and infection. In this study, we tested the inhibitory effect of Au nanoparticles (AuNPs) on pathogenic growth, biofilm formation and invasion. Interestingly, although the synthesized AuNPs had no significant toxicity to the tested pathogens, Candida albicans and Pseudomonas aeruginosa, the nanoparticles strongly inhibited pathogenic biofilm formation and invasion to dental pulp stem cells (DPSCs). Further investigations revealed that AuNPs abundantly bound to the pathogen cells, which likely contributed to their inhibitory effect on biofilm formation and invasion. Moreover, treatment of AuNPs led to activation of immune response-related genes in DPSCs, which may enhance the activity of host immune system against the pathogens. Zeta potential analysis and polyethylene glycol (PEG)/polyethyleneimine (PEI) coating tests further showed that the interaction between pathogen cells and AuNPs is associated with electrostatic attractions. Our findings shed novel light on the application of nanomaterials in fighting against clinical pathogens, and imply that the traditional growth inhibition test is not the only way to evaluate the drug effect during the screening of antimicrobial agents.

The role of microbes and their antimicrobial susceptibilities in both acute and chronic infections of the dental pulp in humans has been well studied. Presently, no data are available on endodontic pathogens in cheetahs (Acinonyx jubatus). The aim of this study was to isolate and identify the bacteria found in the canine teeth of cheetahs, where the pulp was necrotic and exposed due to a complicated crown fracture. Thirty-six microbiologic samples were taken from root canals (RCs) of the canine teeth of 19 cheetahs: one pulp sample was taken from 10 cheetahs, four samples from 2 cheetahs, two samples from 3 cheetahs, and three samples from 4 cheetahs. Exposed pulps were cultured for aerobic and anaerobic bacteria; an additional screening with a 16S rRNA-specific polymerase chain reaction (PCR) was used for the last six samples. Antimicrobial susceptibility of isolates was determined by use of the Kirby-Bauer diffusion test. In total, 59 cultivable isolates belonging to 19 microbial species and 13 genera were recovered from the 36 RCs sampled. Only two samples yielded no cultivable bacteria. Thirty-two (54.49%) of the cultivable isolates were Gram positive and 27 (45.71%) were Gram negative. The maximum number of isolates cultivated from an individual RC was six. Facultative anaerobes (62.72%) were the most common bacteria of the RCs that yielded cultivable bacteria. Of the isolates, 28.81% were aerobic and 8.47% were strict anaerobes. The antimicrobials that showed the greatest efficacy in vitro against the different bacteria isolates were amikacin and gentamicin. The more common bacterial species isolated by PCR were anaerobes (60.8%), facultative anaerobes (30.2%), and aerobes (8.6%).

The management of occlusal caries still remains a major challenge for researchers as well as for general practitioners. The present paper reviews and discusses the most up-to-date knowledge and evidence of the biological principles guiding diagnosis, risk assessment, and management of the caries process on occlusal surfaces. In addition, it considers the whole spectrum of the caries process on occlusal surfaces, ranging from the molecular ecology of occlusal biofilms to the management of deep occlusal caries lesions. Studies using molecular methods with focus on biofilms in relation to occlusal caries should explore the relationship between the function and the structural composition of these biofilms to understand the role of occlusal biofilms in caries development. State-of-the-art measures to evaluate risk for occlusal caries lesion activity, caries incidence, and progression should include the assessment of the occlusal biofilm and the stage of tooth eruption. Careful clinical examination of non-cavitated lesions, including assessment of the lesion activity status, remains the major tool to determine the immediate treatment need and to follow on the non-operative treatment outcome. Even medium occlusal caries lesions in the permanent dentition may be treated by non-invasive fissure sealing. By extending the criteria for non-invasive treatments, traditional restoration of occlusal surfaces can be postponed or even avoided, and the dental health in children and adolescents can be improved. Selective removal (incomplete) to soft dentin in deep carious lesions has greater success rates than stepwise excavation. Selective (complete) removal to firm dentin has a lower success rate due to increased pulp exposure., (© 2016 S. Karger AG, Basel.)

Objectives: This study examines the wound healing process in exposed rat pulp when capped with experimental adhesive resin systems., Methods: Experimental adhesive resin system for direct pulp capping was composed of primer-I (PI), -II (PII), and -III (PIII) and an experimental bonding agent (EBA). PI was Clearfil(®) SE Bond(®)/Primer (CSP) containing 5.0 wt% CaCl2, PII was PI containing 10 wt% nanofiller (Aerosil(®) 380), and PIII was CSP containing 5.0 wt% of compounds of equal moles of synthetic peptides (pA and pB) derived from dentin matrix protein 1. EBA was Clearfil(®) SE Bond(®)/Bond (CSB) containing 10 wt% hydroxyapatite powders. Three experimental groups were designed. PI was assigned to experimental Groups 1 and 3. PII was assigned to experimental Groups 2 and 3. PIII and EBA were assigned to all experimental adhesive groups. Control teeth were capped with calcium hydroxide preparation (Dycal(®)), and CSP and CSB were applied to the cavity. The rats were sacrificed after each observation period (14, 28, 56, and 112 days). The following parameters were evaluated: pulp tissue disorganization, inflammatory cell infiltration, reparative dentin formation (RDF), and bacterial penetration., Results: There were no significant differences among all the groups for all parameters and all observation periods (p>0.05, Kruskal-Wallis test). All groups showed initial RDF at 14 days postoperatively and extensive RDF until 112 days postoperatively. Groups 2 and 3 demonstrated higher quantity of mineralized dentin bridge formation compared with Group 1., Conclusions: Addition of nanofillers to the primer was effective in promoting high-density RDF., Clinical Significance: Experimentally developed adhesive resin systems induce the exposed pulp to produce almost the same quantity of reparative dentin as calcium hydroxide. However, we need further studies to elucidate whether the same results could be obtained in humans., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objectives: To investigate the effect of various dilutions of antibiotic medicaments used in endodontic regeneration on the survival of human dental pulp stem cells (DPSCs) and to determine their antibacterial effect against established Enterococcus faecalis biofilm., Materials and Methods: The cytotoxic and antibacterial effects of different triple (TAP) and double antibiotic paste (DAP) dilutions (0.125, 0.25, 0.5, 1, and 10 mg/ml) were tested against Enterococcus faecalis established biofilm and DPSC. Established bacterial biofilm were exposed to antibiotic dilutions for 3 days. Then, biofilms were collected, spiral plated, and the numbers of bacterial colony forming units (CFU/ml) were determined. For the cytotoxic effect, lactate dehydrogenase activity assays (LDH) and cell viability assays (WST-1) were used to measure the percentage of DPSC cytotoxicity after 3-day treatment with the same antibiotic dilutions. A general linear mixed model was used for statistical analyses (α = 0.05)., Results: All antibiotic dilutions significantly decreased the bacterial CFU/ml. For WST-1 assays, all antibiotic dilutions except 0.125 mg/ml significantly reduced the viability of DPSC. For LDH assays, the three lowest tested concentrations of DAP (0.5, 0.25, 0.125 mg/ml) and the two lowest concentrations of TAP (0.25 and 0.125 mg/ml) were non-toxic to DPSC., Conclusions: All tested dilutions had an antibacterial effect against E. faecalis. However, 0.125 mg/ml of DAP and TAP showed a significant antibacterial effect with no cytotoxic effects on DPSCs., Clinical Relevance: Using appropriate antibiotic concentrations of intracanal medicament during endodontic regeneration procedures is critical to disinfect root canal and decrease the adverse effects on stem cells.

Photoactivated disinfection has a strong local antimicrobial effect. In the field of dentistry it is an emerging adjunct to mechanical debridement during endodontic and periodontal treatment. In the present study, we investigate the effect of photoactivated disinfection using riboflavin as a photosensitizer and blue LED light for activation, and compare it to photoactivated disinfection with the widely used combination of toluidine blue O and red light. Riboflavin is highly biocompatible and can be activated with LED lamps at hand in the dental office. To date, no reports are available on the antimicrobial effect of photoactivated disinfection using riboflavin/blue light on oral microorganisms. Planktonic cultures of eight organisms frequently isolated from periodontal and/or endodontic lesions (Aggregatibacter actinomycetemcomitans, Candida albicans, Enterococcus faecalis, Escherischia coli, Lactobacillus paracasei, Porphyromonas gingivalis, Prevotella intermedia and Propionibacterium acnes) were subjected to photoactivated disinfection with riboflavin/blue light and toluidine blue O/red light, and survival rates were determined by CFU counts. Within the limited irradiation time of one minute, photoactivated disinfection with riboflavin/blue light only resulted in minor reductions in CFU counts, whereas full kills were achieved for all organisms when using toluidine blue O/red light. The black pigmented anaerobes P. gingivalis and P. intermedia were eradicated completely by riboflavin/blue light, but also by blue light treatment alone, suggesting that endogenous chromophores acted as photosensitizers in these bacteria. On the basis of our results, riboflavin cannot be recommended as a photosensitizer used for photoactivated disinfection of periodontal or endodontic infections.

Background: Paleomicrobiology is a special branch of micropaleontology concerned with the study of bacterial fossils. We have used the term 'oral paleomicrobiology', as in this review we have focused on the ancient oral microflora. Recently, dental calculus and dental pulp have been identified as rich sources of ancient microbial DNA. Study of this ancient genetic material opens a new door to the ancient world. This review gives an overview of history of ancient DNA research, various techniques of analyzing ancient DNA in dental calculus and dental pulp, and the implications of the oral paleomicrobiology., Materials and Methods: A comprehensive literature search was performed in the following databases-pubmed, medline and google scholar for studies published before 10 April, 2015. The following keywords were used- 'ancient DNA', 'ancient oral flora, 'oral paleomicrobiology' and 'oral microbiome', '16S rRNA sequencing'. To obtain additional data, a manual search was performed using the reference lists of selected articles., Result: As a result of literature search, 27 articles were found in pubmed, 12 in google scholar and one in medline. Eight more articles were selected from the reference list of selected articles., Conclusion: The combination of microbiology and paleontology has brought a revolution in the study of human evolution and microbial communities. The naturally well-preserved samples of microbial DNA from dental pulp and microbial colonies trapped in dental calculus are a potential source of microbial genetic material, which will prove invaluable in resolving mysteries of the past. This may be a beginning of a new era of oral paleomicrobiology, which will contribute in our studies about prevention of disease by establishing symbiosis between human beings and their microbiome.