Journal of Dental Research

Inflammation is triggered by stimulation of innate sensors that recognize pathogens, chemical and physical irritants, and damaged cells subsequently initiating a well-orchestrated adaptive immune response. Immune cell activation is a strictly regulated and self-resolving process supported by an array of negative feedback mechanisms to sustain tissue homeostasis. The disruption of these regulatory pathways forms the basis of chronic inflammatory diseases, including periodontitis. Ubiquitination, a covalent posttranslational modification of target proteins with ubiquitin, has a profound effect on the stability and activity of its substrates, thereby regulating the immune system at molecular and cellular levels. Through the cooperative actions of E3 ubiquitin ligases and deubiquitinases, ubiquitin modifications are implicated in several biological processes, including proteasomal degradation, transcriptional regulation, regulation of protein-protein interactions, endocytosis, autophagy, DNA repair, and cell cycle regulation. A20 (tumor necrosis factor α–induced protein 3 or TNFAIP3) is a ubiquitin-editing enzyme that mainly functions as an endogenous regulator of inflammation through termination of nuclear factor (NF)–κB activation as part of a negative feedback loop. A20 interacts with substrates that reside downstream of immune sensors, including Toll-like receptors, nucleotide-binding oligomerization domain-containing receptors, lymphocyte receptors, and cytokine receptors. Due to its pleiotropic functions as a ubiquitin binding protein, deubiquitinase and ubiquitin ligase, and its versatile role in various signaling pathways, aberrant A20 levels are associated with numerous conditions such as rheumatoid arthritis, diabetes, systemic lupus erythematosus, inflammatory bowel disease, psoriasis, Sjögren syndrome, coronary artery disease, multiple sclerosis, cystic fibrosis, asthma, cancer, neurological disorders, and aging-related sequelae. Similarly, A20 has recently been implicated as an essential regulator of inflammation in the oral cavity. This review presents information on the ubiquitin system and regulation of NF-κB by ubiquitination using A20 as a representative molecule and highlights how the dysregulation of this system can lead to several immune pathologies, including oral cavity–related disorders mainly focusing on periodontitis.

Osseointegrated dental implants are a revolutionary tool in the armament of reconstructive dentistry, employed to replace missing teeth and restore masticatory, occlusal, and esthetic functions. Like natural teeth, the orally exposed part of dental implants offers a pristine nonshedding surface for salivary pellicle-mediated microbial adhesion and biofilm formation. In early colonization stages, these bacterial communities closely resemble those of healthy periodontal sites, with lower diversity. Because the peri-implant tissues are more susceptible to endogenous oral infections, understanding of the ecological triggers that underpin the microbial pathogenesis of peri-implantitis is central to developing improved prevention, diagnosis, and therapeutic strategies. The advent of next-generation sequencing (NGS) technologies, notably applied to 16S ribosomal RNA gene amplicons, has enabled the comprehensive taxonomic characterization of peri-implant bacterial communities in health and disease, revealing a differentially abundant microbiota between these 2 states, or with periodontitis. With that, the peri-implant niche is highlighted as a distinct ecosystem that shapes its individual resident microbial community. Shifts from health to disease include an increase in diversity and a gradual depletion of commensals, along with an enrichment of classical and emerging periodontal pathogens. Metatranscriptomic profiling revealed similarities in the virulence characteristics of microbial communities from peri-implantitis and periodontitis, nonetheless with some distinctive pathways and interbacterial networks. Deeper functional assessment of the physiology and virulence of the well-characterized microbial communities of the peri-implant niche will elucidate further the etiopathogenic mechanisms and drivers of the disease.

Periodontal and periapical lesions are infectious inflammatory osteolitytic conditions in which a complex inflammatory immune response mediates bone destruction. However, the uncertainty of a lesion’s progressive or stable phenotype complicates understanding of the cellular and molecular mechanisms triggering lesion activity. Evidence from clinical and preclinical studies of both periodontal and periapical lesions points to a high receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) ratio as the primary determinant of osteolytic activity, while a low RANKL/OPG ratio is often observed in inactive lesions. Proinflammatory cytokines directly modulate RANKL/OPG expression and consequently drive lesion progression, along with pro-osteoclastogenic support provided by Th1, Th17, and B cells. Conversely, the cooperative action between Th2 and Tregs subsets creates an anti-inflammatory and proreparative milieu associated with lesion stability. Interestingly, the trigger for lesion status switch from active to inactive can originate from an unanticipated RANKL immunoregulatory feedback, involving the induction of Tregs and a host response outcome with immunological tolerance features. In this context, dendritic cells (DCs) appear as potential determinants of host response switch, since RANKL imprint a tolerogenic phenotype in DCs, described to be involved in both Tregs and immunological tolerance generation. The tolerance state systemically and locally suppresses the development of exacerbated and pathogenic responses and contributes to lesions stability. However, immunological tolerance break by comorbidities or dysbiosis could explain lesions relapse toward activity. Therefore, this article will provide a critical review of the current knowledge concerning periodontal and periapical lesions activity and the underlying molecular mechanisms associated with the host response. Further studies are required to unravel the role of immunological responsiveness or tolerance in the determination of lesion status, as well as the potential cooperative and/or inhibitory interplay among effector cells and their impact on RANKL/OPG balance and lesion outcome.

Periodontitis has been independently associated with the chronic noncommunicable diseases that most frequently lead to death worldwide. The aim of the present systematic review was to study whether people with periodontitis/edentulism are at increased risk of all-cause and cause-specific mortality as compared with those without periodontitis/edentulism. Cohort studies were included that 1) evaluated periodontitis or edentulism as exposures in relation to all-cause or cause-specific mortality as an outcome and 2) reported effect estimates as hazard ratios, risk ratios, or odds ratios with 95% CIs or crude numbers. Two review authors independently searched for eligible studies, screened the titles and abstracts, did full-text analysis, extracted the data from the published reports, and performed the risk-of-bias assessment. In case of disagreement, a third review author was consulted. Study results were summarized through random effects meta-analyses. A total of 57 studies were included, involving 48 cohorts and 5.71 million participants. Periodontitis was associated with increased risk of all-cause mortality (risk ratio, 1.46 [95% CI, 1.15 to 1.85]) and mortality due to cardiovascular diseases (1.47 [1.14 to 1.90]), cancer (1.38 [1.24 to 1.53]), coronary heart disease (2.58 [2.20 to 3.03]), cerebrovascular diseases (3.11 [2.42 to 3.98]), but not pneumonia (0.98 [0.69 to 1.38]). Edentulism (all types) was associated with increased risk of all-cause mortality (1.66 [1.46 to 1.88]) and mortality due to cardiovascular diseases (2.03 [1.50 to 2.74]), cancer (1.55 [1.24 to 1.94]), pneumonia (1.72 [1.07 to 2.78]), coronary heart disease (2.98 [2.43 to 3.65]), and cerebrovascular diseases (3.18 [2.24 to 4.51]). Periodontitis and its ultimate sequela (edentulism) are associated with an increased risk of all-cause and cause-specific mortality (PROSPERO CRD42018100095).

Unemployment rates in the United States are rapidly increasing as a result of the COVID-19 pandemic and attendant economic disruption. As employees lose their jobs, many will lose their employer-sponsored dental insurance (ESDI). Changes in insurance coverage are directly related to the oral health of the population, with many at risk of losing access to dental care. We assessed the impact of recent unemployment rates on insurance coverage and dental utilization. We estimated changes in dental insurance coverage at the state level, using previously applied econometric estimates. Expected changes in types of dental procedures performed at dental practices nationwide were assessed using a microsimulation model, using national practice survey data. Changes in emergency department (ED) visits for dental problems were estimated by fitting trendlines to ED visit patterns by payer type. Sensitivity analyses were conducted to assess how variations in unemployment rates and rates of ESDI in response to unemployment could alter the results. Since March 2020, the national unemployment rate has increased by 8.40 percentage points, an increase expected to result in more than 16 million individuals losing ESDI in the United States. Of these individuals, 45.0% are likely to enroll in their state’s Medicaid and Children’s Health Insurance Program, and 47.0% are expected to become uninsured. With these expected changes in dental insurance coverage, the average dental practice would experience decreases in routine checkup visits but increases in tooth extraction, a procedure that is highly used by publicly insured or uninsured patients. In addition, dental-related ED visits would be expected to grow by 4.0%. Losses of employment caused by the COVID-19 in the United States can have countervailing effects on people’s health by impeding access to dental care. Lack of dental insurance is expected to be more pronounced in states that have not expanded Medicaid or do not provide Medicaid dental benefits for adults.

Oral microbiomes vary in cariogenic potential; these differences may be established early in life. A major concern is whether mothers transmit cariogenic bacteria to their children. Here we characterize early salivary microbiome development and the potential associations of that development with route of delivery, breastfeeding, and mother’s oral health, and we evaluate transmission of microbes between mother and child. We analyzed saliva and metadata from the Center for Oral Health Research in Appalachia. For this cohort study, we sequenced the V6 region of the 16S rRNA gene and used quantitative polymerase chain reaction to detect Streptococcus mitis, Streptococcus sobrinus, Streptococcus mutans, Streptococcus oralis, and Candida albicans in the saliva from mothers and their infants, collected at 2, 9, and 12 mo (Pennsylvania site) and 2, 12, and 24 mo (West Virginia site). Breastfed children had lower relative abundances of Prevotella and Veillonella. If mothers had decayed, missing, or filled teeth, children had greater abundances of Veillonella and Actinomyces. There was little evidence of maternal transmission of selected microbes. At 12 mo, children’s microbiomes were more similar to other children’s than to their mothers’. Infants’ salivary microbiomes became more adult-like with age but still differed with mothers’ microbiomes at 12 mo. There was little evidence supporting transmission of selected microbes from mothers to children, but risk of colonization was associated with tooth emergence. Children are likely to acquire cariogenic bacteria from a variety of sources, including foods and contact with other children and adults.

This 24-mo randomized controlled trial was based on a double-blind parallel design, and it compared the effectiveness of 2 fluoride application protocols in arresting dentine caries in primary teeth. Three-year-old children with active dentine caries were recruited and randomly allocated to 2 treatment groups. Children in group A received a semiannual application of a 25% silver nitrate (AgNO₃) solution followed by a commercially available varnish with 5% sodium fluoride (NaF) on the carious tooth surfaces. Children in group B received a semiannual application of a 25% AgNO₃ solution followed by another commercially available varnish with 5% NaF containing functionalized tricalcium phosphate (fTCP). Carious tooth surfaces that were hard when probing were classified as arrested. Intention-to-treat analysis and a hierarchical generalized linear model were undertaken. A total of 408 children with 1,831 tooth surfaces with active dentine caries were recruited at baseline, and 356 children (87%) with 1,607 tooth surfaces (88%) were assessed after 24 mo. At the 24-mo evaluation, the mean (SD) number of arrested carious tooth surfaces per child were 1.8 (2.2) and 2.6 (3.3) for group A (without fTCP) and group B (with fTCP), respectively (P = 0.003). The arrest rates at the tooth surface level were 42% for group A and 57% for group B (P < 0.001). Results of the hierarchical generalized linear model indicated that protocol B (with fTCP) had a higher predicted probability (PP = 0.656) in arresting dentine caries than protocol A (without fTCP; PP = 0.500) when the carious lesions were on buccal/lingual surfaces, were on anterior teeth, had dental plaque coverage, and were in children from low-income families (P = 0.046). In conclusion, protocol B, which applied a 25% AgNO₃ solution followed by a commercially available 5% NaF varnish with fTCP semiannually, is more effective in arresting dentine caries in primary teeth as compared with protocol A, which applied a 25% AgNO₃ solution followed by another commercially available 5% NaF varnish without fTCP semiannually (ClinicalTrials.gov NCT03423797).

Candida albicans is known to form polymicrobial biofilms with various Streptococcus spp., including mitis and mutans group streptococci. Streptococcus gordonii (mitis group) has been shown to bind avidly to C. albicans hyphae via direct cell-to-cell interaction, while the cariogenic pathogen Streptococcus mutans (mutans group) interacts with the fungal cells via extracellular glucans. However, the biophysical properties of these cross-kingdom interactions at the single-cell level during the early stage of biofilm formation remain understudied. Here, we examined the binding forces between S. mutans (or S. gordonii) and C. albicans in the presence and absence of in situ glucans on the fungal surface using single-cell atomic force microscopy and their influence on biofilm initiation and subsequent development under cariogenic conditions. The data show that S. gordonii binding force to the C. albicans surface is significantly higher than that ofS. mutans to the fungal surface (~2-fold). However, S. mutans binding forces are dramatically enhanced when the C. albicans cell surface is locally coated with extracellular glucans (~6-fold vs. uncoated C. albicans), which vastly exceeds the forces between S. gordonii andC. albicans. The enhanced binding affinity of S. mutans to glucan-coated C. albicans resulted in a larger structure during early biofilm initiation compared to S. gordonii–C. albicans biofilms. Ultimately, this resulted in S. mutans dominance composition in the 3-species biofilm model under cariogenic conditions. This study provides a novel biophysical aspect of Candida-streptococcal interaction whereby extracellular glucans may selectively favor S. mutans binding interactions with C. albicans during cariogenic biofilm development.

Biofilm-mediated oral diseases such as dental caries and periodontal disease remain highly prevalent in populations worldwide. Biofilm formation initiates with the attachment of primary colonizers onto surfaces, and in the context of caries, the adhesion of oral streptococci to dentinal collagen is crucial for biofilm progression. It is known that dentinal collagen suffers from glucose-associated crosslinking as a function of aging or disease; however, the effect of collagen crosslinking on the early adhesion and subsequent biofilm formation of relevant oral streptococci remains unknown. Therefore, the aim of this work was to determine the impact of collagen glycation on the initial adhesion of primary colonizers such as Streptococcus mutans UA159 and Streptococcus sanguinis SK 36, as well as its effect on the early stages of streptococcal biofilm formation in vitro. Type I collagen matrices were crosslinked with either glucose or methylglyoxal. Atomic force microscopy nanocharacterization revealed morphologic and mechanical changes within the collagen matrix as a function of crosslinking, such as a significantly increased elastic modulus in crosslinked fibrils. Increased nanoadhesion forces were observed for S. mutans on crosslinked collagen surfaces as compared with the control, and retraction curves obtained for both streptococcal strains demonstrated nanoscale unbinding behavior consistent with bacterial adhesin-substrate coupling. Overall, glucose-crosslinked substrates specifically promoted the initial adhesion, biofilm formation, and insoluble extracellular polysaccharide production of S. mutans, while methylglyoxal treatment reduced biofilm formation for both strains. Changes in the adhesion behavior and biofilm formation of oral streptococci as a function of collagen glycation could help explain the biofilm dysbiosis seen in older people and patients with diabetes. Further studies are necessary to determine the influence of collagen crosslinking on the balance between acidogenic and nonacidogenic streptococci to aid in the development of novel preventive and therapeutic treatment against dental caries in these patients.

To investigate the characteristics and molecular events of dental pulp stem cells (DPSCs) for tissue regeneration with aging, we isolated and analyzed the stem cells from human exfoliated deciduous teeth (SHED) and permanent teeth of young (Y-DPSCs) and old (A-DPSCs) adults. Results showed that the stemness and osteogenic differentiation capacity of DPSCs decreased with aging. The RNA sequencing results showed that glycine, serine, and threonine metabolism was one of the most enriched gene clusters among SHED, Y-DPSCs, and A-DPSCs, according to analysis based on the Kyoto Encyclopedia of Genes and Genomes. The expression of serine metabolism–related enzymes phosphoserine aminotransferase 1 (PSAT1) and phosphoglycerate (PHGDH) decreased in A-DPSCs and provided less methyl donor S-adenosylmethionine (SAM) for DNA methylation, leading to the hypomethylation of the senescence marker p16 (CDNK2A). Furthermore, the proliferation and differentiation capacity of Y-DPSCs and SHED decreased after PHGDH siRNA treatment, which reduced the level of SAM. Convincingly, the ratios of PSAT1-, PHGDH-, or proliferating cell nuclear antigen–positive cells in the dental pulp of old permanent teeth were less than those in the dental pulp of deciduous teeth and young permanent teeth. In summary, the stemness and differentiation capacity of DPSCs decreased with aging. The decreased serine metabolism in A-DPSCs upregulated the expression of p16 via attenuating its DNA methylation, resulting in DPSC aging. Our finding indicated that serine metabolism and 1 carbon unit participated in stem cell aging, which provided new direction for stem cell aging study and intervention.

Calcitonin gene-related peptide (CGRP) plays a crucial role in the modulation of orofacial pain, and we hypothesized that CGRP mediated a neuron-glia crosstalk in orofacial pain. The objective of this study was to elucidate the mechanisms whereby CGRP mediated trigeminal neuron-glia crosstalk in modulating orofacial pain. Orofacial pain was elicited by ligating closed-coil springs between incisors and molars. Trigeminal neurons and satellite glial cells (SGCs) were cultured for mechanistic exploration. Gene and protein expression were determined through immunostaining, polymerase chain reaction, and Western blot. Orofacial pain was evaluated through the rat grimace scale. Our results revealed that the expressions of CGRP were elevated in both trigeminal neurons and SGCs following the induction of orofacial pain. Intraganglionic administration of CGRP and olcegepant exacerbated and alleviated orofacial pain, respectively. The knockdown of CGRP through viral vector-mediated RNA interference was able to downregulate CGRP expressions in both neurons and SGCs and to alleviate orofacial pain. CGRP upregulated the expression of inducible nitric oxide synthase through the p38 signaling pathway in cultured SGCs. In turn, L-arginine (nitric oxide donor) was able to enhance orofacial pain by upregulating CGRP expressions in vivo. In cultured trigeminal neurons, L-arginine upregulated the expression of CGRP, and this effect was diminished by cilnidipine (N-type calcium channel blocker) while not by mibefradil (L-type calcium channel blocker). In conclusion, CGRP modulated orofacial pain through upregulating the expression of nitric oxide through the p38 signaling pathway in SGCs, and the resulting nitric oxide in turn stimulated CGRP expression through N-type calcium channel in neurons, building a CGRP-mediated positive-feedback neuron-glia crosstalk.