Journal of Dental Research

The speech of the AADOCR President-Elect Alexandre R. Vieira given at the opening ceremony of the 52nd Annual Meeting and Exhibition in Portland, March 15, 2023.

Vaccine technology has evolved continuously since its beginning, and mucosal vaccination, including intranasal, sublingual, and oral administrations, has recently gained great scientific interest. The oral mucosa represents a promising minimally invasive route for antigen delivery, mainly at sublingual and buccal mucosal sites, and it is easily accessible, immunologically rich, and able to promote an effective systemic and local immune response. The aim of this review is to provide an updated overview on the technologies for oral mucosal vaccination, with emphasis on mucoadhesive biomaterial-based delivery systems. Polymeric-based nanoparticles, multilayer films and wafers, liposomes, microneedles, and thermoresponsive gels are the most investigated strategies to deliver antigens locally, showing mucoadhesive properties, controlled release of the antigen, and the ability to enhance immunological responses. These formulations have achieved adequate properties in terms of vaccine stability, are minimally invasive, and are easy to produce and manage. To date, oral mucosa vaccine delivery systems represent a promising and open field of research. Future directions should focus on the role of these systems to induce sustained innate and adaptive immune responses, by integrating the recent advances achieved in mucoadhesion with those related to vaccine technology. Being painless, easy to administer, highly stable, safe, and effective, the antigen delivery systems via the oral mucosa may represent a useful and promising strategy for fast mass vaccination, especially during pandemic outbreaks.

Access to dental clinics is a feature of the neighborhood service environment that may influence oral health care utilization. However, residential selection poses a challenge to causal inference. By studying the involuntary relocation of survivors of the 2011 Great East Japan Earthquake and Tsunami (GEJE), we examined the association between changes in geographic distance to dental clinics and dental visits. Longitudinal data from a cohort of older residents in Iwanuma City directly impacted by the GEJE were analyzed in this study. The baseline survey was conducted in 2010, 7 mo before the occurrence of GEJE, and a follow-up was conducted in 2016. Using Poisson regression models, we estimated the incidence rate ratios (IRR) and 95% confidence intervals (CIs) for the uptake of denture use (as a proxy for dental visits) according to changes in distance from the nearest dental clinic to their house. Age at baseline, housing damage by the disaster, deteriorating economic conditions, and worsened physical activity were used as confounders. Among the 1,098 participants who had not worn dentures before the GEJE, 495 were men (45.1%), with a mean ± SD age at baseline of 74.0 ± 6.9 y. During the 6-year follow-up, 372 (33.9%) participants initiated denture use. Compared to those who experienced a large increase in distance to dental clinics (>370.0–6,299.1 m), a large decrease in distance to dental clinics (>429.0–5,382.6 m) was associated with a marginally significantly higher initiation of denture use among disaster survivors (IRR = 1.28; 95% CI, 0.99–1.66). The experience of major housing damage was independently associated with higher initiation of denture use (IRR = 1.77; 95% CI, 1.47−2.14). Improved geographic access to dental clinics may increase dental visits of disaster survivors. Further studies in non-disaster-affected areas are needed to generalize these findings.

This study aimed to evaluate the efficacy of deep learning (DL) for the identification and classification of various types of dental implant systems (DISs) using a large-scale multicenter data set. We also compared the classification accuracy of DL and dental professionals. The data set, which was collected from 5 college dental hospitals and 10 private dental clinics, contained 37,442 (24.8%) periapical and 113,291 (75.2%) panoramic radiographic images and consisted of a total of 10 manufacturers and 25 different types of DISs. The classification accuracy of DL was evaluated using a pretrained and modified ResNet-50 architecture, and comparison of accuracy performance and reading time between DL and dental professionals was conducted using a self-reported questionnaire. When comparing the accuracy performance for classification of DISs, DL (accuracy: 82.0%; 95% confidence interval [CI], 75.9%–87.0%) outperformed most of the participants (mean accuracy: 23.5% ± 18.5%; 95% CI, 18.5%–32.3%), including dentists specialized (mean accuracy: 43.3% ± 20.4%; 95% CI, 12.7%–56.2%) and not specialized (mean accuracy: 16.8% ± 9.0%; 95% CI, 12.8%–20.9%) in implantology. In addition, DL tends to require lesser reading and classification time (4.5 min) than dentists who specialized (75.6 ± 31.0 min; 95% CI, 13.1–78.4) and did not specialize (91.3 ± 38.3 min; 95% CI, 74.1–108.6) in implantology. DL achieved reliable outcomes in the identification and classification of various types of DISs, and the classification accuracy performance of DL was significantly superior to that of specialized or nonspecialized dental professionals. DL as a decision support aid can be successfully used for the identification and classification of DISs encountered in clinical practice.

Water residue and replacement difficulty cause insufficient adhesive infiltration in demineralized dentin matrix (DDM), which produces a defective hybrid layer and thus a bonding durability problem, severely plaguing adhesive dentistry for decades. In this study, we propose that the unique properties of a highly hydrated interface of the porous DDM can give rise to 1 new type of interface, confined liquid water, which accounts for most of the residue water and may be the main cause of insufficient infiltration. To prove our hypothesis, 3 metal ions with increasing binding affinity and complex stability (Na⁺, Ca²⁺, and Cu²⁺) were introduced respectively to coordinate negatively charged groups such as -PO₄³⁻, -COO⁻ abundant in the DDM interface. Strong chelation of Ca²⁺ and Cu²⁺ rapidly released the confined water, significantly improving penetration of hydrophobic adhesive monomers, while Na⁺ had little effect. A significant decrease of defects in the hybrid layer and a much decreased modulus gap between the hybrid layer and the adhesive layer greatly optimized the microstructure and micromechanical properties of the tooth–resin bonding interface, thus improving the effectiveness and durability of dentin bonding substantially. This study paves the way for a solution to the core scientific issue of contemporary adhesive dentistry: water residue and replacement in dentin bonding, both theoretically and practically.

Dental caries remains the most widespread chronic disease worldwide. Basically, caries originates within biofilms accumulated on dental enamel. Despite the nonrenewable nature of the enamel tissue, targeted preventive strategies are still very limited. We previously introduced customized multifunctional proteinaceous pellicles (coatings) for controlling bacterial attachment and subsequent biofilm succession. Stemmed from our whole proteome/peptidome analysis of the in vivo acquired enamel pellicle, we designed these pellicles using hybrid mixtures of the most abundant and complementary-acting antimicrobial and antifouling proteins/peptides for synergetic suppression of early biofilms. In conjugating these domains synthetically, their bioinhibitory efficacy was remarkably boosted. Herein, we sought to explore the key structure-function relationship of these potent de novo hybridized conjugates in comparison with their individual domains, solely or in physical mixtures. Specifically, we interrelated the following facets: physicochemical and 3-dimensional folding characteristics via molecular dynamics simulations, adopted secondary structure by circular dichroism, immobilization capacity on enamel through high–spatial resolution multiphoton microscopy, and biofilm suppression potency. Our data showed consistent associations among the increased preference for protein folding structures, α-helix content, and enamel-immobilization capacity; all were inversely correlated with the attached bioburden. The expressed phenotypes could be explained by the adopted strongly amphipathic helical conformation upon conjugation, mediated by the highly anionic and acidic N-terminal pentapeptide shared region/motif for enhanced immobilization on enamel. In conclusion, conjugating bioactive proteins/peptides is a novel translational approach to engineer robust antibiofilm pellicles for caries prevention. The adopted α-helical conformation is key to enhance the antibiofilm efficacy and immobilization capacity on enamel that are promoted by certain physicochemical properties of the constituent domains. These data are valuable for bioengineering versatile therapeutics to prevent/arrest dental caries, a condition that otherwise requires invasive treatments with substantial health care expenditures.

Ectopic enrichment of oral microbes in the gut is a notable alteration in gut microbial balance. These microbes are likely delivered from the oral cavity with saliva and food; however, evidence of oral–gut microbial transmission is insufficient and needs further investigation. In this observational study, we examined 144 pairs of saliva and stool samples collected from community-dwelling adults to verify the oral–gut microbial link and identify the relevant influencing factors on the increased abundance of oral microbes within the gut. The bacterial composition of each sample was determined using PacBio single-molecule long-read sequencing of the full-length 16S ribosomal RNA gene and amplicon sequence variant (ASV) analysis. Although the bacterial compositions of salivary and gut microbiota were distinctly different, at least 1 ASV was shared between salivary and gut microbiota in 72.9% of subjects. Shared ASVs accounted for 0.0% to 63.1% (median 0.14%) of the gut microbiota in each subject and frequently included abundant Streptococcus salivarius and Streptococcus parasanguinis. Their total relative abundance in the gut was significantly higher in older subjects or those with dental plaque accumulation. The gut microbiota with ≥5% of shared ASVs displayed a higher abundance of Streptococcus, Lactobacillus, and Klebsiella and a lower abundance of Faecalibacterium, Blautia, Megamonas, and Parabacteroides. Our study presents evidence for the translocation of oral bacteria to the gut in community-dwelling adults and suggests that aging and dental plaque accumulation contribute to an increased abundance of oral microbes in the gut, which might be relevant to the compositional shift in the gut commensals.

Dental caries lesions are a clinical manifestation of disease, preceded by microbial dysbiosis, which is poorly characterized and thought to be associated with saccharolytic taxa. Here, we assessed the associations between the oral microbiome of children and various caries risk factors such as demographics and behavioral and clinical data across early childhood and characterized over time the salivary and dental plaque microbiome of children before clinical diagnosis of caries lesions. Children (N = 266) were examined clinically at ~1, 2.5, 4, and 6.5 y of age. The microbiome samples were collected at 1, 2.5, and 4 y. Caries groups consisted of children who remained caries free (International Caries Detection and Assessment System [ICDAS] = 0) at all time points (CFAT) (n = 50); children diagnosed with caries (ICDAS ≥ 1) at 6.5 y (C6.5), 4 y (C4), or 2.5 y of age (C2.5); and children with early caries or advanced caries lesions at specific time points. Microbial community analyses were performed on zero-radius operational taxonomic units (zOTUs) obtained from V4 of 16S ribosomal RNA gene amplicon sequences. The oral microbiome of the children was affected by various factors, including antibiotic use, demographics, and dietary habits of the children and their caregivers. At all time points, various risk factors explained more of the variation in the dental plaque microbiome than in saliva. At 1 y, composition of saliva of the C4 group differed from that of the CFAT group, while at 2.5 y, this difference was observed only in plaque. At 4 y, multiple salivary and plaque zOTUs of genera Prevotella and Leptotrichia were significantly higher in samples of the C6.5 group than those of the CFAT group. In conclusion, up to 3 y prior to clinical caries detection, the oral microbial communities were already in a state of dysbiosis that was dominated by proteolytic taxa. Plaque discriminated dysbiotic oral ecosystems from healthy ones better than saliva.

B cells play a vital role in the elimination of periodontal pathogens, the regulation of the immune response, and the induction of tissue destruction. However, the role of B cells in the dysfunction of mesenchymal stem cell (MSC) differentiation to osteoblasts in periodontitis (PD) has been poorly studied. Here we show that the frequency of CD45⁻CD105⁺CD73⁺ MSCs in inflamed periodontal tissues is significantly decreased in patients with PD compared with that of healthy controls. CD19⁺ B cells dominate the infiltrated immune cells in periodontal tissues of patients with PD. Besides, B-cell depletion therapy reduces the alveolar bone loss in a ligature-induced murine PD model. B cells from PD mice express a high level of TGF-β1 and inhibit osteoblast differentiation by upregulating p-Smad2/3 expression and downregulating Runx2 expression. The inhibitory effect of PD B cells on osteoblast differentiation is reduced by TGF-β1 neutralization or Smad2/3 inhibitor. Importantly, B-cell–specific knockout of TGF-β1 in PD mice significantly increases the number of CD45⁻CD105⁺Sca1⁺ MSCs, ALP-positive osteoblast activity, and alveolar bone volume but decreases TRAP-positive osteoclast activity compared with that from control littermates. Lastly, CD19⁺CD27⁺CD38⁻ memory B cells dominate the B-cell infiltrates in periodontal tissues from both patients with PD and patients with PD after initial periodontal therapy. Memory B cells in periodontal tissues of patients with PD express a high level of TGF-β1 and inhibit MSC differentiation to osteoblasts. Thus, TGF-β1 produced by B cells may contribute to alveolar bone loss in periodontitis, in part, by suppressing osteoblast activity.

Nano-hydroxyapatite (nHAP) is considered a biocompatible agent that promotes the remineralization of dental hard tissue; however, its antibacterial efficacy is under scientific discussion. Therefore, this investigation aimed to specify the inhibitory effects of disaggregated nano-hydroxyapatite (DnHAP) on regrown biofilms and demineralization. Regrown biofilm models of single-species (Streptococcus mutans), dual-species (S. mutans and Candida albicans), and saliva-derived microcosm biofilms were established in vitro. Repeat treatment with DnHAP was applied to biofilms. The viability, lactic acid, biofilm structure, biomass, the inhibitory effect of demineralization, and virulence factors’ expression were determined. In addition, the biofilm microbial community was analyzed by 16S ribosomal RNA gene sequencing. DnHAP inhibited metabolism, lactic acid production, biomass, and water-insoluble polysaccharide production (P < 0.05) of regrown single/dual-species biofilms. Concerning the saliva-derived biofilms, samples treated with DnHAP showed lower biofilm metabolic activity without significant differences from samples treated with sterile deionized water (P > 0.05); in addition, saliva-derived biofilms treated with DnHAP exhibited lower lactic acid production (P < 0.05). The demineralization of bovine enamel was the lowest in the DnHAP group, as detected by transverse microradiography, and the lesion depth and volume decreased significantly (P < 0.05). The application of DnHAP did not change the diversity of regrown saliva-derived microcosm biofilms. In conclusion, this investigation showed that DnHAP could be a promising solution for the management of regrown biofilms to combat dental caries.

Growing evidence demonstrates the relationship between periodontitis and atherosclerotic cardiovascular diseases. The periodontal pathogen Porphyromonas gingivalis (Pg) has been shown to contribute to the progression of atherosclerosis. Cyclic diadenylate monophosphate (c-di-AMP) has been widely studied as an immune adjuvant for tumor immunotherapy, given its ability to activate the stimulator of interferon genes (STING) and regulate trained immunity. This study sought to elucidate the role of c-di-AMP in Pg-associated atherosclerosis. Periodontitis and atherosclerosis mouse models were established by ligature application around maxillary second molars and feeding ApoE knockout mice with a high-fat diet. We found that periodontitis and atherosclerosis were more severe in mice exposed to Pg than mice that underwent ligature placement only, while prophylactic treatment with c-di-AMP activated trained immunity and elicited significant alleviation of alveolar bone resorption, as well as reduced blood lipid levels and atherosclerotic plaque accumulation. After 3 mo of intervention, c-di-AMP limited the elevation of cytokines interleukin (IL)–6, IL-1β, tumor necrosis factor α, and interferon β; extracellular matrix remodeling enzymes MMP-2 and MMP-9; and adhesion molecules ICAM-1 and VCAM-1 gene expression. The mechanism underlying Pg-aggravated atherosclerosis may be attributed to changes in microbiota composition in oral and aortic plaques and excess inflammatory response, whereas c-di-AMP could prevent the effects of Pg infection due to its potential ability to activate trained immunity and regulate microecological balance. Our findings suggest a positive role of c-di-AMP in alleviating Pg-aggravated atherosclerosis by regulating the immune response and influencing the local microenvironment.

Immune checkpoint molecule PD-1, expressed on the cell surface, impairs antigen-driven activation of T cells and thus plays a critical role in tumorigenesis, progression, and the poor prognosis of oral squamous cell carcinoma (OSCC). In addition, increasing evidence indicates that PD-1 carried on small extracellular vesicles (sEVs) also mediates tumor immunity, although their contributions to OSCC are yet unclear. Here, we investigated the biological functions of sEV PD-1 in patients with OSCC. The cell cycle, proliferation, apoptosis, migration, and invasion of CAL27 cell lines treated with or without sEV PD-1 were examined in vitro. We performed mass spectrometry to investigate the underlying biological process, combined with an immunohistochemical study of SCC7-bearing mice models and OSCC patient samples. In vitro data demonstrated that sEV PD-1 induced senescence and subsequent epithelial-mesenchymal transition (EMT) in CAL27 cells by ligating with tumor cell surface PD-L1 and activating the p38 mitogen-activated protein kinase (MAPK) pathway. Comprehensive immunohistochemical analysis of the xenograft mice models and OSCC patient samples revealed a very close correlation between the level of circulating sEV PD-1 and lymph node metastasis. These results demonstrate that circulating sEV PD-1 triggers senescence-initiated EMT in a PD-L1-p38 MAPK-dependent manner, contributing to tumor metastasis. It also suggests that the inhibition of sEV PD-1 may be a promising therapeutic target for the treatment of OSCC.

The single-nucleotide polymorphism (SNP) rs2235371 (IRF6 V274I) is associated with nonsyndromic cleft lip with or without cleft palate (NSCL/P) in Han Chinese and other populations but appears to be without a functional effect. To find the common etiologic variant or variants within the haplotype tagged by rs2235371, we carried out targeted sequencing of an interval containing IRF6 in 159 Han Chinese with NSCL/P. This study revealed that the SNP rs12403599, within the IRF6 promoter, is associated with all phenotypes of NSCL/P, especially nonsyndromic cleft lip (NSCLO) and a subphenotype of it, microform cleft lip (MCL). This association was replicated in 2 additional much larger cohorts of cases and controls from the Han Chinese. Conditional logistic analysis indicated that association of rs2235371 with NSCL/P was lost if rs12403599 was excluded. rs12403599 contributes the most risk to MCL: its G allele is responsible for 38.47% of the genetic contribution to MCL, and the odds ratios of G/C and G/G genotypes were 2.91 and 6.58, respectively, for MCL. To test if rs12403599 is functional, we carried out reporter assays in a fetal oral epithelium cells (GMSM-K). Unexpectedly, the risk allele G yielded higher promoter activity in GMSM-K. Consistent with the reporter studies, expression of IRF6 in lip tissues from NSCLO and MCL patients with the G/G phenotype was higher than in those from patients with the C/C phenotype. These results indicate that rs12403599 is tagging the risk haplotype for NSCL/P better than rs2235371 in Han Chinese and supports investigation of the mechanisms by which the allele of rs12403599 affects IRF6 expression and tests of this association in different populations.

Subclinical hypothyroidism (SCH) has been shown to be associated with microbiota. However, the association between SCH and oral microbiota has not yet been elucidated. The results of our previous clinical studies showed that Prevotella intermedia was abundant in the oral microbiota of SCH patients. This study aimed to investigate the relationship between SCH and oral microbiota, verify the pathogenicity of P. intermedia in SCH, and preliminarily explore the possible mechanism. The SCH mouse model with oral application of P. intermedia was established, and the variance in the mouse oral microbiota and changes in thyroid function and metabolism were detected in mice. Student’s t test and analysis of variance were used for statistical analysis. Oral application of P. intermedia changed the composition of the oral microbiota of SCH mice, which enhanced the damage to the thyroid and decreased the expression of functional genes of the thyroid. Moreover, P. intermedia decreased oxygen consumption and aggravated glucose and lipid metabolism disorders in SCH mice. Glucose tolerance and insulin tolerance decreased, and the triglyceride content of the liver and inflammatory infiltration in adipose tissue increased in SCH mice after P. intermedia stimulation. Mechanistically, P. intermedia increased the proportion of CD4⁺ T cells in cervical lymph nodes and thyroids in SCH mice. Th1 cells were suggested to play an important role in the pathogenesis of SCH involving P. intermedia. In conclusion, P. intermedia aggravated SCH manifestations, including thyroid dysfunction and glucose and lipid metabolism disorders, by causing immune imbalance in mice. This study sheds new light on the pathogenesis of SCH from the perspective of oral microbiota.

The enamel knot (EK), located at the center of cap stage tooth germs, is a transitory cluster of nondividing epithelial cells. The EK acts as a signaling center that provides positional information for tooth morphogenesis and regulates the growth of tooth cusps. To identify species-specific cuspal patterns, this study analyzed the cellular mechanisms in the EK that were related to bone morphogenetic protein (Bmp), which plays a crucial role in cell proliferation and apoptosis. To understand the cellular mechanisms in the EK, the differences between 2 species showing different cuspal patterning, mouse (pointy bunodont cusp) and gerbil (flat lophodont cusp), were analyzed with quantitative reverse transcriptase polymerase chain reaction and immunofluorescent staining. Based on these, we performed protein-soaked bead implantation on tooth germs of the 2 different EK regions and compared the cellular behavior in the EKs of the 2 species. Many genes related with cell cycle, cell apoptosis, and cell proliferation were involved in BMP signaling in the EK during tooth development. A comparison of the cell proliferation and apoptosis associated with Bmp revealed distinctive patterns of the cellular mechanisms. Our findings indicate that the cellular mechanisms, such as cell proliferation and apoptosis, in the EK are related to Bmp4 and play an important role in tooth morphogenesis.