Molecular profiling of the vestibular lamina highlights a key role for Hedgehog signalling.

The vestibular lamina (VL) forms the oral vestibule, creating a gap between the teeth, lips and cheeks. In a number of ciliopathies, formation of the vestibule is defective, leading to the creation of multiple frenula. In contrast to the neighbouring dental lamina, which forms the teeth, little is known about the genes that pattern the VL. Here, we establish a molecular signature for the usually non-odontogenic VL in mice and highlight several genes and signalling pathways that may play a role in its development. For one of these, the Sonic hedgehog (Shh) pathway, we show that co-receptors Gas1, Cdon and Boc are highly expressed in the VL and act to enhance the Shh signal from the forming incisor region. In Gas1 mutant mice, expression of Gli1 was disrupted and the VL epithelium failed to extend due to a loss of proliferation. This defect was exacerbated in Boc/Gas1 double mutants and could be phenocopied using cyclopamine in culture. Signals from the forming teeth, therefore, control development of the VL, coordinating the development of the dentition and the oral cavity.

Changing perspectives on early hominin diets.

It is axiomatic that knowledge of the diets of extinct hominin species is central to any understanding of their ecology and our evolution. The importance of diet in the paleontological realm has led to the employment of multiple approaches in its elucidation. Some of these have deep historical roots, while others are dependent upon more recent technical and methodological advances. Historically, studies of tooth size, shape, and structure have been the gold standard for reconstructing diet. They focus on species-level adaptations, and as such, they can set theoretical brackets for dietary capabilities within the context of specific evolutionary moments. Other methods (e.g., analyses of dental calculus, biogeochemistry, and dental microwear) have only been developed within the past few decades, but are now beginning to yield evidence of the actual foods consumed by individuals represented by fossil remains. Here we begin by looking at these more "direct" forms of evidence of diet before showing that, when used in conjunction with other techniques, these "multi-proxy" approaches can raise questions about traditional interpretations of early hominin diets and change the nature of paleobiological interpretations.

Resilience of the replacing dentition in adult reptiles.

The dentition is critical to animal survival and teeth are present in modern vertebrates including teleost fish, sharks, amphibians, mammals and reptiles. The developmental processes that give rise to teeth are not just preserved through evolution but also share high level of similarity with the embryogenesis of other ectodermal organs. In this review we go beyond the embryonic phase of tooth development to life-long tooth replacement. We will address the origins of successional teeth, the location of putative tissue-resident stem cells, how de novo tooth formation continues throughout life and how teeth are shed in a spatially and temporally controlled manner. We review the evidence that the dental epithelium, which is the earliest recognizable dental structure in the reptilian dentition, serves as a putative niche for tissue-resident epithelial stem cells and recent molecular findings from transcriptomics carried out in reptilian dentitions. We discuss how odontoclasts resorb the primary tooth allowing eruption of the successional tooth. The reptiles, particularly lizards, are emerging as some of the most accessible animals to study tooth replacement which has relevance to evolution of the dentition and human dental disorders.

Circadian clock genes REV-ERBα regulates the secretion of IL-1ß in deciduous tooth pulp stem cells by regulating autophagy in the process of physiological root resorption of deciduous teeth.

Physiological root resorption is a common occurrence during the development of deciduous teeth in children. Previous research has shown that the regulation of the inflammatory microenvironment through autophagy in DDPSCs is a significant factor in this process. However, it remains unclear why there are variations in the autophagic status of DDPSCs at different stages of physiological root resorption. To address this gap in knowledge, this study examines the relationship between the circadian clock of DDPSCs, the autophagic status, and the periodicity of masticatory behavior. Samples were collected from deciduous teeth at various stages of physiological root resorption, and DDPSCs were isolated and cultured for analysis. The results indicate that the circadian rhythm of important autophagy genes, such as Beclin-1 and LC3, and the clock gene REV-ERBα in DDPSCs, disappears under mechanical stress. Additionally, the study found that REV-ERBα can regulate Beclin-1 and LC3. Evidence suggests that mechanical stress is a trigger for the regulation of autophagy via REV-ERBα. Overall, this study highlights the importance of mechanical stress in regulating autophagy of DDPSCs via REV-ERBα, which affects the formation of the inflammatory microenvironment and plays a critical role in physiological root resorption in deciduous teeth.

Metabolomics for dental caries diagnosis: Past, present, and future.

Dental caries, a prevalent global infectious condition affecting over 95% of adults, remains elusive in its precise etiology. Addressing the complex dynamics of caries demands a thorough exploration of taxonomic, potential, active, and encoded functions within the oral ecosystem. Metabolomic profiling emerges as a crucial tool, offering immediate insights into microecosystem physiology and linking directly to the phenotype. Identified metabolites, indicative of caries status, play a pivotal role in unraveling the metabolic processes underlying the disease. Despite challenges in metabolite variability, the use of metabolomics, particularly via mass spectrometry and nuclear magnetic resonance spectroscopy, holds promise in caries research. This review comprehensively examines metabolomics in caries prevention, diagnosis, and treatment, highlighting distinct metabolite expression patterns and their associations with disease-related bacterial communities. Pioneering in approach, it integrates singular and combinatory metabolomics methodologies, diverse biofluids, and study designs, critically evaluating prior limitations while offering expert insights for future investigations. By synthesizing existing knowledge, this review significantly advances our comprehension of caries, providing a foundation for improved prevention and treatment strategies.

Nicotinamide Riboside Modulates HIF-1 Signaling to Maintain and Enhance Odontoblastic Differentiation in Human Dental Pulp Stem Cells.

Human dental pulp stem cells (hDPSCs) play a vital role in the regeneration of the pulp-dentin complex after pulp disease. While the regeneration efficiency relies on the odontoblastic differentiation capacity of hDPSCs, this is difficult to regulate within the pulp cavity. Although nicotinamide riboside (NR) has been found to promote tissue regeneration, its specific role in pulp-dentin complex regeneration is not fully understood. Here, we aimed to explore the role of NR in the odontoblastic differentiation of hDPSCs and its underlying molecular mechanism. It was found that NR enhanced the viability and retarded senescence in hDPSCs with higher NAD+/NADH levels. In contrast to the sustained action of NR, the multi-directional differentiation of hDPSCs was enhanced after NR pre-treatment. Moreover, in an ectopic pulp regeneration assay in nude mice, transplantation of hDPSCs pretreated with NR promoted the formation of a dentin-like structure surrounded by cells positively expressing DMP-1 and DSPP. RNA-Seq demonstrated inhibition of the HIF-1 signaling pathway in hDPSCs pretreated with NR. The number of HIF-1α-positive cells was significantly decreased in hDPSCs pretreated by NR in vivo. Similarly, NR significantly downregulated the expression of HIF-1α in vitro. The findings suggested that NR could potentially regulate hDPSC odontoblastic differentiation and promote the development of innovative strategies for dental pulp repair.

Single-cell RNA sequencing reveals vascularization-associated cell subpopulations in dental pulp: PDGFRß+ DPSCs with activated PI3K/AKT pathway.

BACKGROUND: This study aims to address challenges in dental pulp regeneration therapy. The heterogeneity of DPSCs poses challenges, especially in stem cell transplantation for clinical use, particularly when sourced from donors of different ages and conditions. METHODS: Pseudotime analysis was employed to analyze single-cell sequencing data, and immunohistochemical studies were conducted to investigate the expression of fibronectin 1 (FN1). We performed in vitro sorting of PDGFRß+ DPSCs using flow cytometry. A series of functional assays, including cell proliferation, scratch, and tube formation assays, were performed to experimentally validate the vasculogenic capabilities of the identified PDGFRß+ DPSC subset. Furthermore, gene-edited mouse models were utilized to demonstrate the importance of PDGFRß+ DPSCs. Transcriptomic sequencing was conducted to compare the differences between PDGFRß+ DPSCs and P1-DPSCs. RESULTS: Single-cell sequencing analysis unveiled a distinct subset, PDGFRß+ DPSCs, characterized by significantly elevated FN1 expression during dental pulp development. Subsequent cell experiments demonstrated that this subset possesses remarkable abilities to promote HUVEC proliferation, migration, and tube formation. Gene-edited mouse models confirmed the vital role of PDGFRß+ DPSCs in dental pulp development. Transcriptomic sequencing and in vitro experiments demonstrated that the PDGFR/PI3K/AKT signaling pathway is a crucial factor mediating the proliferation rate and pro-angiogenic properties of PDGFRß+ DPSCs. CONCLUSION: We defined a new subset, PDGFRß+ DPSCs, characterized by strong proliferative activity and pro-angiogenic capabilities, demonstrating significant clinical translational potential.

S100a6 knockdown promotes the differentiation of dental epithelial cells toward the epidermal lineage instead of the odontogenic lineage.

Tooth development is a complex process involving various signaling pathways and genes. Recent findings suggest that ion channels and transporters, including the S100 family of calcium-binding proteins, may be involved in tooth formation. However, our knowledge in this regard is limited. Therefore, this study aimed to investigate the expression of S100 family members and their functions during tooth formation. Tooth germs were extracted from the embryonic and post-natal mice and the expression of S100a6 was examined. Additionally, the effects of S100a6 knockdown and calcium treatment on S100a6 expression and the proliferation of SF2 cells were examined. Microarrays and single-cell RNA-sequencing indicated that S100a6 was highly expressed in ameloblasts. Immunostaining of mouse tooth germs showed that S100a6 was expressed in ameloblasts but not in the undifferentiated dental epithelium. Additionally, S100a6 was localized to the calcification-forming side in enamel-forming ameloblasts. Moreover, siRNA-mediated S100a6 knockdown in ameloblasts reduced intracellular calcium concentration and the expression of ameloblast marker genes, indicating that S100a6 is associated with ameloblast differentiation. Furthermore, S100a6 knockdown inhibited the ERK/PI3K signaling pathway, suppressed ameloblast proliferation, and promoted the differentiation of the dental epithelium toward epidermal lineage. Conclusively, S100a6 knockdown in the dental epithelium suppresses cell proliferation via calcium and intracellular signaling and promotes differentiation of the dental epithelium toward the epidermal lineage.

The Ruminant sorting mechanism protects teeth from abrasives.

Dental wear due to ingestion of dust and grit has deleterious consequences. Herbivores that could not wash their food hence had to evolve particularly durable teeth, in parallel to the evolution of dental chewing surface complexity to increase chewing efficacy. The rumen sorting mechanism increases chewing efficacy beyond that reached by any other mammal and has been hypothesized to also offer an internal washing mechanism, which would be an outstanding example of an additional advantage by a physiological adaptation, but in vivo evidence is lacking so far. Here, we investigated four cannulated, live cows that received a diet to which sand was added. Silica in swallowed food and feces reflected experimental dietary sand contamination, whereas the regurgitate submitted to rumination remained close to the silica levels of the basal food. This helps explain how ruminants are able to tolerate high levels of dust or grit in their diet, with less high-crowned teeth than nonruminants in the same habitat. Palaeo-reconstructions based on dental morphology and dental wear traces need to take the ruminants' wear-protection mechanism into account. The inadvertent advantage likely contributed to the ruminants' current success in terms of species diversity.

Dental data challenge the ubiquitous presence of Homo in the Cradle of Humankind.

The origins of Homo, as well as the diversity and biogeographic distribution of early Homo species, remain critical outstanding issues in paleoanthropology. Debates about the recognition of early Homo, first appearance dates, and taxonomic diversity within Homo are particularly important for determining the role that southern African taxa may have played in the origins of the genus. The correct identification of Homo remains also has implications for reconstructing phylogenetic relationships between species of Australopithecus and Paranthropus, and the links between early Homo species and Homo erectus. We use microcomputed tomography and landmark-free deformation-based three-dimensional geometric morphometrics to extract taxonomically informative data from the internal structure of postcanine teeth attributed to Early Pleistocene Homo in the southern African hominin-bearing sites of Sterkfontein, Swartkrans, Drimolen, and Kromdraai B. Our results indicate that, from our sample of 23 specimens, only 4 are unambiguously attributed to Homo, 3 of them coming from Swartkrans member 1 (SK 27, SK 847, and SKX 21204) and 1 from Sterkfontein (Sts 9). Three other specimens from Sterkfontein (StW 80 and 81, SE 1508, and StW 669) approximate the Homo condition in terms of overall enamel-dentine junction shape, but retain Australopithecus-like dental traits, and their generic status remains unclear. The other specimens, including SK 15, present a dominant australopith dental signature. In light of these results, previous dietary and ecological interpretations can be reevaluated, showing that the geochemical signal of one tooth from Kromdraai (KB 5223) and two from Swartkrans (SK 96 and SKX 268) is consistent with that of australopiths.

Single-cell RNA-seq analysis of rat molars reveals cell identity and driver genes associated with dental mesenchymal cell differentiation.

BACKGROUND: The molecular mechanisms and signaling pathways involved in tooth morphogenesis have been the research focus in the fields of tooth and bone development. However, the cell population in molars at the late bell stage and the mechanisms of hard tissue formation and mineralization remain limited knowledge. RESULTS: Here, we used the rat mandibular first and second molars as models to perform single-cell RNA sequencing (scRNA-seq) analysis to investigate cell identity and driver genes related to dental mesenchymal cell differentiation during the late bell hard tissue formation stage. We identified seven main cell types and investigated the heterogeneity of mesenchymal cells. Subsequently, we identified novel cell marker genes, including Pclo in dental follicle cells, Wnt10a in pre-odontoblasts, Fst and Igfbp2 in periodontal ligament cells, and validated the expression of Igfbp3 in the apical pulp. The dynamic model revealed three differentiation trajectories within mesenchymal cells, originating from two types of dental follicle cells and apical pulp cells. Apical pulp cell differentiation is associated with the genes Ptn and Satb2, while dental follicle cell differentiation is associated with the genes Tnc, Vim, Slc26a7, and Fgfr1. Cluster-specific regulons were analyzed by pySCENIC. In addition, the odontogenic function of driver gene TNC was verified in the odontoblastic differentiation of human dental pulp stem cells. The expression of osteoclast differentiation factors was found to be increased in macrophages of the mandibular first molar. CONCLUSIONS: Our results revealed the cell heterogeneity of molars in the late bell stage and identified driver genes associated with dental mesenchymal cell differentiation. These findings provide potential targets for diagnosing dental hard tissue diseases and tooth regeneration.

Glycerol metabolism contributes to competition by oral streptococci through production of hydrogen peroxide.

As a biological byproduct from both humans and microbes, glycerol's contribution to microbial homeostasis in the oral cavity remains understudied. In this study, we examined glycerol metabolism by Streptococcus sanguinis, a commensal associated with oral health. Genetic mutants of glucose-PTS enzyme II (manL), glycerol metabolism (glp and dha pathways), and transcriptional regulators were characterized with regard to glycerol catabolism, growth, production of hydrogen peroxide (H2O2), transcription, and competition with Streptococcus mutans. Biochemical assays identified the glp pathway as a novel source for H2O2 production by S. sanguinis that is independent of pyruvate oxidase (SpxB). Genetic analysis indicated that the glp pathway requires glycerol and a transcriptional regulator, GlpR, for expression and is negatively regulated by PTS, but not the catabolite control protein, CcpA. Conversely, deletion of either manL or ccpA increased the expression of spxB and a second, H2O2-non-producing glycerol metabolic pathway (dha), indicative of a mode of regulation consistent with conventional carbon catabolite repression (CCR). In a plate-based antagonism assay and competition assays performed with planktonic and biofilm-grown cells, glycerol greatly benefited the competitive fitness of S. sanguinis against S. mutans. The glp pathway appears to be conserved in several commensal streptococci and actively expressed in caries-free plaque samples. Our study suggests that glycerol metabolism plays a more significant role in the ecology of the oral cavity than previously understood. Commensal streptococci, though not able to use glycerol as a sole carbohydrate source for growth, benefit from the catabolism of glycerol through production of both ATP and H2O2. IMPORTANCE: Glycerol is an abundant carbohydrate in the oral cavity. However, little is understood regarding the metabolism of glycerol by commensal streptococci, some of the most abundant oral bacteria. This was in part because most streptococci cannot grow on glycerol as the sole carbon source. In this study, we show that Streptococcus sanguinis, a commensal associated with dental health, can degrade glycerol for persistence and competition through two pathways, one of which generates hydrogen peroxide at levels capable of inhibiting Streptococcus mutans. Preliminary studies suggest that several additional commensal streptococci are also able to catabolize glycerol, and glycerol-related genes are actively expressed in human dental plaque samples. Our findings reveal the potential of glycerol to significantly impact microbial homeostasis, which warrants further exploration.

Non-coding RNAs in endodontic disease.

The immunocompetence and regeneration potential of the dental pulp and its surrounding apical tissues have been investigated extensively in the field of endodontics. While research on the role of non-coding RNAs in these tissues is still in its infancy, it is envisioned that improved understanding of the regulatory function of ncRNAs in pulpal and periapical immune response will help prevent or treat endodontic disease. Of particular importance is the role of these RNAs in regenerating the dentin-pulp complex. In this review, we highlight recent progress on the role of non-coding RNAs in the immune response to endodontic infection as well as the repair and regenerative response to injury.

Automated High-Throughput Biological Sex Identification from Archeological Human Dental Enamel Using Targeted Proteomics.

Biological sex is key information for archeological and forensic studies, which can be determined by proteomics. However, the lack of a standardized approach for fast and accurate sex identification currently limits the reach of proteomics applications. Here, we introduce a streamlined mass spectrometry (MS)-based workflow for the determination of biological sex using human dental enamel. Our approach builds on a minimally invasive sampling strategy by acid etching, a rapid online liquid chromatography (LC) gradient coupled to a high-resolution parallel reaction monitoring (PRM) assay allowing for a throughput of 200 samples per day (SPD) with high quantitative performance enabling confident identification of both males and females. Additionally, we developed a streamlined data analysis pipeline and integrated it into a Shiny interface for ease of use. The method was first developed and optimized using modern teeth and then validated in an independent set of deciduous teeth of known sex. Finally, the assay was successfully applied to archeological material, enabling the analysis of over 300 individuals. We demonstrate unprecedented performance and scalability, speeding up MS analysis by 10-fold compared to conventional proteomics-based sex identification methods. This work paves the way for large-scale archeological or forensic studies enabling the investigation of entire populations rather than focusing on individual high-profile specimens. Data are available via ProteomeXchange with the identifier PXD049326.

Macrophage M2 polarization promotes pulpal inflammation resolution during orthodontic tooth movement.

Mechanical force induces hypoxia in the pulpal area by compressing the apical blood vessels of the pulp, triggering pulpal inflammation during orthodontic tooth movement. However, this inflammation tends to be restorable. Macrophages are recognized as pivotal immunoreactive cells in the dental pulp. Whether they are involved in the resolution of pulpal inflammation in orthodontic teeth remains unclear. In this study, we investigated macrophage polarization and its effects during orthodontic tooth movement. It was demonstrated that macrophages within the dental pulp polarized to M2 type and actively participated in the process of pulpal inflammation resolution. Inflammatory reactions were generated and vascularization occurred in the pulp during orthodontic tooth movement. Macrophages in orthodontic pulp show a tendency to polarize towards M2 type as a result of pulpal hypoxia. Furthermore, by blocking M2 polarization, we found that macrophage M2 polarization inhibits dental pulp-secreting inflammatory factors and enhances VEGF production. In conclusion, our findings suggest that macrophages promote pulpal inflammation resolution by enhancing M2 polarization and maintaining dental health during orthodontic tooth movement.

Nerve growth factor promote osteogenic differentiation of dental pulp stem cells through MEK/ERK signalling pathways.

Nerve growth factor (NGF) and its receptor, tropomyosin receptor kinase A (TrkA), are known to play important roles in the immune and nervous system. However, the effects of NGF on the osteogenic differentiation of dental pulp stem cells (DPSCs) remain unclear. This study aimed to investigate the role of NGF on the osteogenic differentiation of DPSCs in vitro and the underlying mechanisms. DPSCs were cultured in osteogenic differentiation medium containing NGF (50 ng/mL) for 7 days. Then osteogenic-related genes and protein markers were analysed using qRT-PCR and Western blot, respectively. Furthermore, addition of NGF inhibitor and small interfering RNA (siRNA) transfection experiments were used to elucidate the molecular signalling pathway responsible for the process. NGF increased osteogenic differentiation of DPSCs significantly compared with DPSCs cultured in an osteogenic-inducing medium. The NGF inhibitor Ro 08-2750 (10 µM) and siRNA-mediated gene silencing of NGF receptor, TrkA and ERK signalling pathways inhibitor U0126 (10 µM) suppressed osteogenic-related genes and protein markers on DPSCs. Furthermore, our data revealed that NGF-upregulated osteogenic differentiation of DPSCs may be associated with the activation of MEK/ERK signalling pathways via TrkA. Collectively, NGF was capable of promoting osteogenic differentiation of DPSCs through MEK/ERK signalling pathways, which may enhance the DPSCs-mediated bone tissue regeneration.

Wolf-Hirschhorn syndrome candidate 1 (Whsc1) methyltransferase signals via a Pitx2-miR-23/24 axis to effect tooth development.

Wolf-Hirschhorn syndrome (WHS) is a developmental disorder attributed to a partial deletion on the short arm of chromosome 4. WHS patients suffer from oral manifestations including cleft lip and palate, hypodontia, and taurodontism. WHS candidate 1 (WHSC1) gene is a H3K36-specific methyltransferase that is deleted in every reported case of WHS. Mutation in this gene also results in tooth anomalies in patients. However, the correlation between genetic abnormalities and the tooth anomalies has remained controversial. In our study, we aimed to clarify the role of WHSC1 in tooth development. We profiled the Whsc1 expression pattern during mouse incisor and molar development by immunofluorescence staining and found Whsc1 expression is reduced as tooth development proceeds. Using real-time quantitative reverse transcription PCR, Western blot, chromatin immunoprecipitation, and luciferase assays, we determined that Whsc1 and Pitx2, the initial transcription factor involved in tooth development, positively and reciprocally regulate each other through their gene promoters. miRNAs are known to regulate gene expression posttranscriptionally during development. We previously reported miR-23a/b and miR-24-1/2 were highly expressed in the mature tooth germ. Interestingly, we demonstrate here that these two miRs directly target Whsc1 and repress its expression. Additionally, this miR cluster is also negatively regulated by Pitx2. We show the expression of these two miRs and Whsc1 are inversely correlated during mouse mandibular development. Taken together, our results provide new insights into the potential role of Whsc1 in regulating tooth development and a possible molecular mechanism underlying the dental defects in WHS.

SNHG1 knockdown promotes osteogenic differentiation of hDFSCs through anti-oxidative stress mediated by autophagy.

The long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) plays a crucial role in tumorigenesis and is frequently employed as a prognostic biomarker. However, its involvement in the osteogenic differentiation of oral stem cells, particularly human dental follicle stem cells (hDFSCs), remains unclear. Our investigation revealed that the absence of SNHG1 enhances the osteogenic differentiation of hDFSCs. Furthermore, the downregulation of SNHG1 induces autophagy in hDFSCs, leading to a reduction in intracellular oxidative stress levels. Notably, this effect is orchestrated through the epigenetic regulation of EZH2. Our study unveils a novel function of SNHG1 in governing the osteogenic differentiation of hDFSCs, offering fresh insights for an in-depth exploration of the molecular mechanisms underlying dental follicle development. These findings not only provide a foundation for advancing the understanding of SNHG1 but also present innovative perspectives for promoting the repair and regeneration of periodontal supporting tissue, ultimately contributing to the restoration of periodontal health and tooth function.

Nondental Invasive Procedures and Risk of Infective Endocarditis: Time for a Revisit: A Science Advisory From the American Heart Association.

There have been no published prospective randomized clinical trials that have: (1) established an association between invasive dental and nondental invasive procedures and risk of infective endocarditis; or (2) defined the efficacy and safety of antibiotic prophylaxis administered in the setting of invasive procedures in the prevention of infective endocarditis in high-risk patients. Moreover, previous observational studies that examined the association of nondental invasive procedures with the risk of infective endocarditis have been limited by inadequate sample size. They have typically focused on a few potential at-risk surgical and nonsurgical invasive procedures. However, recent investigations from Sweden and England that used nationwide databases and demonstrated an association between nondental invasive procedures, and the subsequent development of infective endocarditis (in particular, in high-risk patients with infective endocarditis) prompted the development of the current science advisory.

Dental Caries, Race and Incident Ischemic Stroke, Coronary Heart Disease, and Death.

BACKGROUND: Dental caries is a highly prevalent disease worldwide. In the United States, untreated dental caries is present in >1 in 5 adults. The objective of this study was to determine the relationship between dental caries and incident ischemic stroke, coronary heart disease (CHD) events, and death. METHODS: The dental cohort (n=6351) of the ARIC study (Atherosclerosis Risk in Communities) was followed for incident ischemic stroke, CHD event, and all-cause mortality. Of all the participants at visit 4 (n=11 656), those who were unable to go through dental examination, or with prevalent ischemic stroke and CHD events, were excluded. The full-mouth dental examination was conducted at visit 4 (1996-1998), assessing dental caries. The dose response of decayed, missing, and filled surfaces due to caries was assessed and related to the outcome. Outcomes were assessed through the end of 2019. Additionally, the effect of regular dental care utilization on dental caries was evaluated. RESULTS: Participants with ≥1 dental caries had an increased risk of stroke (adjusted hazard ratio [HR], 1.40 [95% CI, 1.10-1.79]) and death (adjusted HR, 1.13 [95% CI, 1.01-1.26]) but not for CHD events (adjusted HR, 1.13 [95% CI, 0.93-1.37]). The association of dental caries and ischemic incident stroke was significantly higher in the African American population compared with the White subgroup (interaction term P=0.0001). Increasing decayed, missing, and filled surfaces were significantly associated with stroke (adjusted HR, 1.006 [95% CI, 1.001-1.011]) and death (adjusted HR, 1.003 [95% CI, 1.001-1.005]) but not CHD (adjusted HR, 1.002 [95% CI, 1.000-1.005]). Regular dental care utilization lowered (adjusted odds ratio, 0.19 [95% CI, 0.16-0.22]; P<0.001) the chance of caries. CONCLUSIONS: Among the cohort, dental caries was independently associated with the risk of ischemic stroke and death, with the effect higher in African American participants. Regular dental care utilization was associated with a lower chance of caries, emphasizing its relevance in the prevention of these events.