Fungi and bacteria occupy distinct spatial niches within carious dentin.

The role of bacteria in the etiology of dental caries is long established, while the role of fungi has only recently gained more attention. The microbial invasion of dentin in advanced caries especially merits additional research. We evaluated the fungal and bacterial community composition and spatial distribution within carious dentin. Amplicon 16S rRNA gene sequencing together with quantitative PCR was used to profile bacterial and fungal species in caries-free children (n = 43) and 4 stages of caries progression from children with severe early childhood caries (n = 32). Additionally, healthy (n = 10) and carious (n = 10) primary teeth were decalcified, sectioned, and stained with Grocott's methenamine silver, periodic acid Schiff (PAS) and calcofluor white (CW) for fungi. Immunolocalization was also performed using antibodies against fungal ß-D-glucan, gram-positive bacterial lipoteichoic acid, gram-negative endotoxin, Streptococcus mutans, and Candida albicans. We also performed field emission scanning electron microscopy (FESEM) to visualize fungi and bacteria within carious dentinal tubules. Bacterial communities observed included a high abundance of S. mutans and the Veillonella parvula group, as expected. There was a higher ratio of fungi to bacteria in dentin-involved lesions compared to less severe lesions with frequent preponderance of C. albicans, C. dubliniensis, and in one case C. tropicalis. Grocott's silver, PAS, CW and immunohistochemistry (IHC) demonstrated the presence of fungi within carious dentinal tubules. Multiplex IHC revealed that fungi, gram-negative, and gram-positive bacteria primarily occupied separate dentinal tubules, with rare instances of colocalization. Similar findings were observed with multiplex immunofluorescence using anti-S. mutans and anti-C. albicans antibodies. Electron microscopy showed monomorphic bacterial and fungal biofilms within distinct dentin tubules. We demonstrate a previously unrecognized phenomenon in which fungi and bacteria occupy distinct spatial niches within carious dentin and seldom co-colonize. The potential significance of this phenomenon in caries progression warrants further exploration.

Proteomic Analyses Discern the Developmental Inclusion of Albumin in Pig Enamel: A New Model for Human Enamel Hypomineralization.

Excess albumin in enamel is a characteristic of the prevalent developmental dental defect known as chalky teeth or molar hypomineralization (MH). This study uses proteomic analyses of pig teeth to discern between developmental origin and post-eruptive contamination and to assess the similarity to hypomineralized human enamel. Here, the objective is to address the urgent need for an animal model to uncover the etiology of MH and to improve treatment. Porcine enamel is chalky and soft at eruption; yet, it hardens quickly to form a hard surface and then resembles human teeth with demarcated enamel opacities. Proteomic analyses of enamel from erupted teeth, serum, and saliva from pigs aged 4 (n = 3) and 8 weeks (n = 2) and human (n = 4) molars with demarcated enamel opacities show alpha-fetoprotein (AFP). AFP expression is limited to pre- and perinatal development and its presence in enamel indicates pre- or perinatal inclusion. In contrast, albumin is expressed after birth, indicating postnatal inclusion into enamel. Peptides were extracted from enamel and analyzed by nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) after tryptic digestion. The mean total protein number was 337 in the enamel of all teeth with 13 different unique tryptic peptides of porcine AFP in all enamel samples but none in saliva samples. Similarities in the composition, micro-hardness, and microstructure underscore the usefulness of the porcine model to uncover the MH etiology, cellular mechanisms of albumin inclusion, and treatment for demarcated opacities.

Pit-and-fissure sealants on primary molars are a cost savings.

BACKGROUND: In this study, the authors examine the cost-effectiveness of light-polymerized resin-based fluoride sealants on primary molars in high caries risk children younger than 6 years. METHODS: The authors examined the cost-effectiveness of pit-and-fissure sealant (PFS) treatment on primary molars by comparing sealed and unsealed molars treated in the outpatient clinic or operating room. Using 1,884 primary molars followed over a 5-year period, the authors used a mixed-effects regression model to estimate the probability of caries development. They used restricted means to estimate years free of caries for carious molars. They used a decision tree to address uncertainty due to PFS treatment failure, predict the expected value associated with each strategy, and estimate the incremental cost-effectiveness ratio using a 3% discount rate to adjust future cost and outcomes to present value. RESULTS: Over 5 years, the cost of care was $90 for unsealed molars and $75 for sealed molars. Unsealed molars remained caries free for 4.32 years compared with 4.85 years in sealed molars. The cost-effectiveness of PFS treatment was dominant, leading to a savings of $25 for each caries-free year gained and overall savings of $742 million for the United States dental health system over a 5-year period. CONCLUSIONS: PFS treatment is associated with cost savings and a delay in caries development and should be considered in children with high caries risk. PRACTICAL IMPLICATIONS: Policy makers should consider reimbursement of PFS treatment on primary molars in high caries risk children.

The Predominant Oral Microbiota Is Acquired Early in an Organized Pattern.

The human oral cavity is sterile prior to birth, and we have limited knowledge of how complex oral communities are assembled. To examine bacterial acquisition and community assembly over the first year of life, oral samples from a cohort of nine infants and their mothers were collected, and bacterial community composition was studied by 16S rRNA gene sequencing. Exogenous species including skin and environmental bacteria were present initially, but were quickly replaced by a small, shared microbial community of species common to all infants and adults. Subsequent ordered microbial succession and the formation of increasingly complex communities was observed. By one year of age oral microbial community composition converged to a profile that was remarkably similar among children. The introduction of new nutrient sources, but not tooth eruption, was associated with increasing complexity. Infants had fewer species than mothers, mostly accounted for by the lack of certain anaerobes, and showing that the acquisition and assembly of oral microbial communities continues past infancy. When relative abundance was considered, a shared set of species accounted for the majority of the microbial community at all ages, indicating that the dominant structure of the oral microbiome establishes early, and suggesting that it persists throughout life.

Sealed primary molars are less likely to develop caries.

BACKGROUND: The authors examined the association between light-polymerized resin-based fluoride-releasing sealants and the development of pit-and-fissure caries on primary molars. METHODS: In this 3-year retrospective study, the authors reviewed the dental records of 297 children (1,352 teeth) younger than 6 years who were at high caries risk. Sealant placement or nonplacement on primary molars in the outpatient clinic and operating room setting was recorded, and random-effects logistic regression analysis accounting for the effect of data clustering was performed to measure caries incidence over time. RESULTS: The odds of developing pit-and-fissure carious lesions on sealed primary molars were 0.055 times (95% confidence interval [CI], 0.011 to 0.285; P = .001) and 0.013 times (95% CI, 0.001 to 0.159; P = .001) the odds of that on nonsealed primary molars in the outpatient clinic and in the operating room, respectively. In molars that became carious, those sealed were associated with longer time to caries development in both the outpatient clinic (2.69 years, 95% CI, 2.08 to 3.29) and operating room (1.97 years, 95% CI, 1.45 to 2.48). CONCLUSIONS: Sealed primary molars were less likely to develop pit-and-fissure caries when placed in both the clinic and operating room settings. PRACTICAL IMPLICATIONS: Dental sealants on primary molars should be considered for children at high caries risk.

Use of Silver Diamine Fluoride for Dental Caries Management in Children and Adolescents, Including Those with Special Health Care Needs.

BACKGROUND: This manuscript presents evidence-based guidance on the use of 38 percent silver diamine fluoride (SDF) for dental caries management in children and adolescents, including those with special health care needs. A guideline workgroup formed by the American Academy of Pediatric Dentistry developed guidance and an evidence-based recommendation regarding the application of 38 percent SDF to arrest cavitated caries lesions in primary teeth. TYPES OF STUDIES REVIEWED: The basis of the guideline's recommendation is evidence from an existing systematic review "Clinical trials of silver diamine fluoride in arresting caries among children: A systematic review." (JDR Clin Transl Res 2016;1[3]:201-10). A systematic search was conducted in PubMed®/MEDLINE, Embase®, Cochrane Central Register of Controlled Trials, and gray literature databases to identify randomized controlled trials and systematic reviews reporting on the effect of silver diamine fluoride and address peripheral issues such as adverse effects and cost. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach was used to assess the quality of the evidence and the evidence-to-decision framework was employed to formulate a recommendation. RESULTS: The panel made a conditional recommendation regarding the use of 38 percent SDF for the arrest of cavitated caries lesions in primary teeth as part of a comprehensive caries management program. After taking into consideration the low cost of the treatment and the disease burden of caries, panel members were confident that the benefits of SDF application in the target populations outweigh its possible undesirable effects. Per GRADE, this is a conditional recommendation based on low-quality evidence. Conclusions and practical implications: The guideline intends to inform the clinical practices involving the application of 38 percent SDF to enhance dental caries management outcomes in children and adolescents, including those with special health care needs. These recommended practices are based upon the best available evidence to-date. A 38 percent SDF protocol is included in Appendix II.

The collection of NFATc1-dependent transcripts in the osteoclast includes numerous genes non-essential to physiologic bone resorption.

Osteoclasts are specialized secretory cells of the myeloid lineage important for normal skeletal homeostasis as well as pathologic conditions of bone including osteoporosis, inflammatory arthritis and cancer metastasis. Differentiation of these multinucleated giant cells from precursors is controlled by the cytokine RANKL, which through its receptor RANK initiates a signaling cascade culminating in the activation of transcriptional regulators which induce the expression of the bone degradation machinery. The transcription factor nuclear factor of activated T-cells c1 (NFATc1) is the master regulator of this process and in its absence osteoclast differentiation is aborted both in vitro and in vivo. Differential mRNA expression analysis by microarray is used to identify genes of potential physiologic relevance across nearly all biologic systems. We compared the gene expression profile of murine wild-type and NFATc1-deficient osteoclast precursors stimulated with RANKL and identified that the majority of the known genes important for osteoclastic bone resorption require NFATc1 for induction. Here, five novel RANKL-induced, NFATc1-dependent transcripts in the osteoclast are described: Nhedc2, Rhoc, Serpind1, Adcy3 and Rab38. Despite reasonable hypotheses for the importance of these molecules in the bone resorption pathway and their dramatic induction during differentiation, the analysis of mice with mutations in these genes failed to reveal a function in osteoclast biology. Compared to littermate controls, none of these mutants demonstrated a skeletal phenotype in vivo or alterations in osteoclast differentiation or function in vitro. These data highlight the need for rigorous validation studies to complement expression profiling results before functional importance can be assigned to highly regulated genes in any biologic process.

Control of bone resorption in mice by Schnurri-3.

Mice lacking the large zinc finger protein Schnurri-3 (Shn3) display increased bone mass, in part, attributable to augmented osteoblastic bone formation. Here, we show that in addition to regulating bone formation, Shn3 indirectly controls bone resorption by osteoclasts in vivo. Although Shn3 plays no cell-intrinsic role in osteoclasts, Shn3-deficient animals show decreased serum markers of bone turnover. Mesenchymal cells lacking Shn3 are defective in promoting osteoclastogenesis in response to selective stimuli, likely attributable to reduced expression of the key osteoclastogenic factor receptor activator of nuclear factor-κB ligand. The bone phenotype of Shn3-deficient mice becomes more pronounced with age, and mice lacking Shn3 are completely resistant to disuse osteopenia, a process that requires functional osteoclasts. Finally, selective deletion of Shn3 in the mesenchymal lineage recapitulates the high bone mass phenotype of global Shn3 KO mice, including reduced osteoclastic bone catabolism in vivo, indicating that Shn3 expression in mesenchymal cells directly controls osteoblastic bone formation and indirectly regulates osteoclastic bone resorption.

NFATc1 in mice represses osteoprotegerin during osteoclastogenesis and dissociates systemic osteopenia from inflammation in cherubism.

Osteoporosis results from an imbalance in skeletal remodeling that favors bone resorption over bone formation. Bone matrix is degraded by osteoclasts, which differentiate from myeloid precursors in response to the cytokine RANKL. To gain insight into the transcriptional regulation of bone resorption during growth and disease, we generated a conditional knockout of the transcription factor nuclear factor of activated T cells c1 (Nfatc1). Deletion of Nfatc1 in young mice resulted in osteopetrosis and inhibition of osteoclastogenesis in vivo and in vitro. Transcriptional profiling revealed NFATc1 as a master regulator of the osteoclast transcriptome, promoting the expression of numerous genes needed for bone resorption. In addition, NFATc1 directly repressed osteoclast progenitor expression of osteoprotegerin, a decoy receptor for RANKL previously thought to be an osteoblast-derived inhibitor of bone resorption. "Cherubism mice", which carry a gain-of-function mutation in SH3-domain binding protein 2 (Sh3bp2), develop osteoporosis and widespread inflammation dependent on the proinflammatory cytokine, TNF-alpha. Interestingly, deletion of Nfatc1 protected cherubism mice from systemic bone loss but did not inhibit inflammation. Taken together, our study demonstrates that NFATc1 is required for remodeling of the growing and adult skeleton and suggests that NFATc1 may be an effective therapeutic target for osteoporosis associated with inflammatory states.

The cementum-dentin junction also contains glycosaminoglycans and collagen fibrils.

The presence of glycosaminoglycans (GAGs) and their contribution to mechanical properties of the cementum-dentin junction (CDJ) were investigated using nanometer scale characterization techniques. Five to two millimeter thick transverse sections from the apical ends of human molars were ultrasectioned at room temperature under wet conditions using a diamond knife and an ultramicrotome. The structure of the CDJ under dry and wet conditions before and after digestion of GAGs and collagen fibrils was studied using an atomic force microscope (AFM). The mechanical properties of the untreated and enzyme treated CDJ under wet conditions were studied using an AFM-based nanoindenter. GAG digestion was performed for 1, 3, and 5 h at 37 degrees C using chondroitinase-ABC. Collagen fibril digestion was performed for 24 and 48 h at 37 degrees C using collagenase. As reported previously, AFM scans of dry untreated CDJ (control) revealed a valley, which transformed into a peak under wet conditions. The height differences relative to cementum and dentin of untreated and treated CDJ were determined by measuring the CDJ profile under dry and wet conditions. The depth of the valley of GAG and collagen-digested CDJ was greater than that of undigested CDJ under dry conditions. The height of the peak of GAG-digested CDJ was significantly higher than that of the undigested CDJ under wet conditions. The collagen-digested CDJ under wet conditions is assumed to form a valley because of the removal of collagen fibrils from the CDJ. However, the depth of the valley was lower compared to the depth under dry conditions. Wet AFM-based nanoindentation showed that the elastic modulus and hardness of control (3.3+/-1.2 and 0.08+/-0.03 GPa) were significantly higher (ANOVA & SNK, P < 0.05) than chondroitinase-ABC treated CDJ (0.9+/-0.4 and 0.02+/-0.004 GPa) and collagenase treated CDJ (1.5+/-0.6 and 0.04+/-0.01 GPa). No significant difference in mechanical properties between chondroitinase-ABC and collagenase treated CDJ was observed. Based on the results it was concluded that the 10-50 microm wide CDJ is a composite that includes, chondroitin-4-sulfate, chondroitin-6-sulfate, and possibly dermatan sulfate, and collagen fibrils. The association of GAGs with the collagen fibrils provides the observed controlled hydration and partially contributes toward the stiffness of the CDJ under wet conditions.