DENTAL ENAMEL FORMATION AND IMPLICATIONS FOR ORAL HEALTH AND DISEASE.

Dental enamel is the hardest and most mineralized tissue in extinct and extant vertebrate species and provides maximum durability that allows teeth to function as weapons and/or tools as well as for food processing. Enamel development and mineralization is an intricate process tightly regulated by cells of the enamel organ called ameloblasts. These heavily polarized cells form a monolayer around the developing enamel tissue and move as a single forming front in specified directions as they lay down a proteinaceous matrix that serves as a template for crystal growth. Ameloblasts maintain intercellular connections creating a semi-permeable barrier that at one end (basal/proximal) receives nutrients and ions from blood vessels, and at the opposite end (secretory/apical/distal) forms extracellular crystals within specified pH conditions. In this unique environment, ameloblasts orchestrate crystal growth via multiple cellular activities including modulating the transport of minerals and ions, pH regulation, proteolysis, and endocytosis. In many vertebrates, the bulk of the enamel tissue volume is first formed and subsequently mineralized by these same cells as they retransform their morphology and function. Cell death by apoptosis and regression are the fates of many ameloblasts following enamel maturation, and what cells remain of the enamel organ are shed during tooth eruption, or are incorporated into the tooth's epithelial attachment to the oral gingiva. In this review, we examine key aspects of dental enamel formation, from its developmental genesis to the ever-increasing wealth of data on the mechanisms mediating ionic transport, as well as the clinical outcomes resulting from abnormal ameloblast function.

FAM20A mutations and transcriptome analyses of dental pulp tissues of enamel renal syndrome.

AIM: Biallelic loss-of-function FAM20A mutations cause amelogenesis imperfecta (AI) type IG, better known as enamel renal syndrome (ERS), characterized by severe enamel hypoplasia, delayed/failed tooth eruption, intrapulpal calcifications, gingival hyperplasia and nephrocalcinosis. FAM20A binds to FAM20C, the Golgi casein kinase (GCK) and potentiates its function to phosphorylate secreted proteins critical for biomineralization. While many FAM20A pathogenic mutations have been reported, the pathogeneses of orodental anomalies in ERS remain to be elucidated. This study aimed to identify disease-causing mutations for patients with ERS phenotypes and to discern the molecular mechanism underlying ERS intrapulpal calcifications. METHODOLOGY: Phenotypic characterization and whole exome analyses were conducted for 8 families and 2 sporadic cases with hypoplastic AI. A minigene assay was performed to investigate the molecular consequences of a FAM20A splice-site variant. RNA sequencing followed by transcription profiling and gene ontology (GO) analyses were carried out for dental pulp tissues of ERS and the control. RESULTS: Biallelic FAM20A mutations were demonstrated for each affected individual, including 7 novel pathogenic variants: c.590-5T>A, c.625T>A (p.Cys209Ser), c.771del (p.Gln258Argfs*28), c.832_835delinsTGTCCGACGGTGTCCGACGGTGTC CA (p.Val278Cysfs*29), c.1232G>A (p.Arg411Gln), c.1297A>G (p.Arg433Gly) and c.1351del (p.Gln451Serfs*4). The c.590-5T>A splice-site mutation caused Exon 3 skipping, which resulted in an in-frame deletion of a unique region of the FAM20A protein, p.(Asp197_Ile214delinsVal). Analyses of differentially expressed genes in ERS pulp tissues demonstrated that genes involved in biomineralization, particularly dentinogenesis, were significantly upregulated, such as DSPP, MMP9, MMP20 and WNT10A. Enrichment analyses indicated overrepresentation of gene sets associated with BMP and SMAD signalling pathways. In contrast, GO terms related to inflammation and axon development were underrepresented. Among BMP signalling genes, BMP agonists GDF7, GDF15, BMP3, BMP8A, BMP8B, BMP4 and BMP6 were upregulated, while BMP antagonists GREM1, BMPER and VWC2 showed decreased expression in ERS dental pulp tissues. CONCLUSIONS: Upregulation of BMP signalling underlies intrapulpal calcifications in ERS. FAM20A plays an essential role in pulp tissue homeostasis and prevention of ectopic mineralization in soft tissues. This critical function probably depends upon MGP (matrix Gla protein), a potent mineralization inhibitor that must be properly phosphorylated by FAM20A-FAM20C kinase complex.

Mouse genetic background influences the dental phenotype.

Dental enamel covers the crown of the vertebrate tooth and is considered to be the hardest tissue in the body. Enamel develops during secretion of an extracellular matrix by ameloblast cells in the tooth germ, prior to eruption of the tooth into the oral cavity. Secreted enamel proteins direct mineralization patterns during the maturation stage of amelogenesis as the tooth prepares to erupt. The amelogenins are the most abundant enamel proteins and are required for normal enamel development. Phenotypic differences were observed between incisors from individual Amelx (amelogenin) null mice that had a mixed 129xC57BL/6J genetic background and between inbred wild-type (WT) mice with different genetic backgrounds (C57BL/6J, C3H/HeJ, FVB/NJ). We hypothesized that this could be due to modifier genes, as human patients with a mutation in an enamel protein gene causing the enamel defect amelogenesis imperfecta (AI) can also have varied appearance of dentitions within a kindred. Enamel density measurements varied for all WT inbred strains midway during incisor development. Enamel thickness varied between some WT strains, and, unexpectedly, dentin density varied extensively between incisors and molars of all WT and Amelx null strains studied. WTFVB/NJ incisors were more similar to those of Amelx null mice than to those of the other WT strains in terms of incisor height/width ratio and pattern of enamel mineralization. Strain-specific differences led to the conclusion that modifier genes may be implicated in determining both normal development and severity of enamel appearance in AI mouse models and may in future studies be related to phenotypic heterogeneity within human AI kindreds reported in the literature.

Digital restorative workflows for developmental dental defects in young patients: A case series.

BACKGROUND: Digital technology is rapidly changing the provision of oral health care, although its adoption for the oral health care of young patients has lagged. The authors describe digitally supported treatment approaches for managing treatment of developmental dental defects in the early permanent dentition. CASE DESCRIPTION: Four adolescent patients with amelogenesis imperfecta received transitional anterior restorations for esthetic and functional rehabilitation using a variety of digital workflows. Combinations of restoration type, materials, and fabrication methods were selected to meet the needs of each patient on the basis of their specific amelogenesis imperfecta phenotype and chief symptoms. These cases highlight the application of digital technology in pediatric and adolescent dentistry for managing the treatment of developmental dental defects. PRACTICAL IMPLICATIONS: Digitally supported restorative approaches, as described in this report, offer broad applicability of materials and techniques directed at treating the complex restorative needs of young patients in the transitional and early permanent dentition.

WNT5A expression in ameloblastoma and its roles in regulating enamel epithelium tumorigenic behaviors.

Odontogenic tumors originate from the remains of migrating enamel epithelium after the completion of normal tooth genesis. These enamel epithelium remnants exhibit the ability to recapitulate the events that occur during tooth formation. Several lines of evidence suggest that aberrance in the signaling pathways similar to the ones that are used during tooth development, including the WNT pathway, might be the cause of odontogenic tumorigenesis and maintenance. In this study we demonstrated that WNT5A expression was intense in both the epithelial component of ameloblastomas, the most common epithelial odontogenic tumor, and in this tumor's likely precursor cell, the enamel epithelium located at the cervical loop of normal developing human tooth buds. Additionally, when WNT5A was overexpressed in enamel epithelium cells (LS-8), the clones expressing high levels of WNT5A (S) exhibited characteristics of tumorigenic cells, including growth factor independence, loss of anchorage dependence, loss of contact inhibition, and tumor formation in immunocompromised mice. Moreover, overexpression of WNT5A drastically increased LS-8 cell migration and actin reorganization when compared with controls. Suppression of endogenous WNT5A in LS-8 cells (AS) greatly impaired their migration and AS cells failed to form significant actin reorganization and membrane protrusion was rarely seen. Taken together, our data indicate that WNT5A signaling is important in modulating tumorigenic behaviors of enamel epithelium cells in ameloblastomas.

Amelogenesis imperfecta: genotype-phenotype studies in 71 families.

Amelogenesis imperfecta (AI) represents hereditary conditions affecting the quality and quantity of enamel. Six genes are known to cause AI (AMELX, ENAM, MMP20, KLK4, FAM83H, and WDR72). Our aim was to determine the distribution of different gene mutations in a large AI population and evaluate phenotype-genotype relationships. Affected and unaffected family members were evaluated clinically and radiographically by one examiner. Genotyping was completed using genomic DNA obtained from blood or saliva. A total of 494 individuals were enrolled, with 430 (224 affected, 202 unaffected, and 4 not definitive) belonging to 71 families with conditions consistent with the diagnosis of AI. Diverse clinical phenotypes were observed (i.e. hypoplastic, hypocalcified, and hypomaturation). Genotyping revealed mutations in all 6 candidate genes. A molecular diagnosis was made in 132 affected individuals (59%) and in 26 of the families (37%). Mutations involved 12 families with FAM83H (46%), 6 families with AMELX (23%), 3 families with ENAM (11%), 2 families with KLK4 and MMP20 (8% for each gene), and 1 family with a WDR72 mutation (4%). Phenotypic variants were associated with allelic FAM83H and AMELX mutations. Two seemingly unrelated families had the same KLK4 mutation. Families affected with AI where candidate gene mutations were not identified could have mutations not identifiable by traditional gene sequencing (e.g. exon deletion) or they could have promoter sequence mutations not evaluated in this study. However, the results suggest that there remain new AI causative genes to be identified.

Regulation of pH During Amelogenesis.

During amelogenesis, extracellular matrix proteins interact with growing hydroxyapatite crystals to create one of the most architecturally complex biological tissues. The process of enamel formation is a unique biomineralizing system characterized first by an increase in crystallite length during the secretory phase of amelogenesis, followed by a vast increase in crystallite width and thickness in the later maturation phase when organic complexes are enzymatically removed. Crystal growth is modulated by changes in the pH of the enamel microenvironment that is critical for proper enamel biomineralization. Whereas the genetic bases for most abnormal enamel phenotypes (amelogenesis imperfecta) are generally associated with mutations to enamel matrix specific genes, mutations to genes involved in pH regulation may result in severely affected enamel structure, highlighting the importance of pH regulation for normal enamel development. This review summarizes the intra- and extracellular mechanisms employed by the enamel-forming cells, ameloblasts, to maintain pH homeostasis and, also, discusses the enamel phenotypes associated with disruptions to genes involved in pH regulation.

Primary failure of eruption and PTH1R: the importance of a genetic diagnosis for orthodontic treatment planning.

INTRODUCTION: Primary failure of eruption (PFE) is characterized by nonsyndromic eruption failure of permanent teeth in the absence of mechanical obstruction. Recent studies support that this dental phenotype is inherited and that mutations in PTH1R genes explain several familial cases of PFE. The objective of our study was to investigate how genetic analysis can be used with clinical diagnostic information for improved orthodontic management of PFE. METHODS: We evaluated a family (n = 12) that segregated an autosomal dominant form of PFE with 5 affected and 7 unaffected persons. Nine available family members (5 male, 4 female) were enrolled and subsequently characterized clinically and genetically. RESULTS: In this family, PFE segregated with a novel mutation in the PTH1R gene. A heterozygous c.1353-1 G>A sequence alteration caused a putative splice-site mutation and skipping of exon 15 that segregated with the PFE phenotype in all affected family members. CONCLUSIONS: A PTH1R mutation is strongly associated with failure of orthodontically assisted eruption or tooth movement and should therefore alert clinicians to treat PFE and ankylosed teeth with similar caution-ie, avoid orthodontic treatment with a continuous archwire.

Classifying ectodermal dysplasias: Incorporating the molecular basis and pathways (Workshop II).

Hereditary conditions are traditionally classified based either on physical/physiological attributes or using the names of the individuals credited with identifying the condition. For the 170 plus conditions classified as ectodermal dysplasias (EDs), both of these nosological systems are used, at times interchangeably. Over the past decade our knowledge of the human genome and the molecular basis of the EDs have greatly expanded providing the impetus to consider alternative classification systems. The incorporation of the molecular basis of hereditary conditions adds important information allowing effective transfer of objective genetic information that can be lacking from traditional classification systems. Molecular information can be added to the nosological system for the EDs through a hierarchical- and domain-based approach that encompasses the condition's name, mode of inheritance, molecular pathway affected, and specific molecular change. As new molecular information becomes available it can be effectively incorporated using this classification approach. Integrating molecular information into the ED classification system, while retaining well-recognized traditional syndrome names, facilitates communication at and between different groups of people including patients, families, health care providers, and researchers.

2008 International Conference on Ectodermal Dysplasias Classification: conference report.

There are many ways to classify ectodermal dysplasia syndromes. Clinicians in practice use a list of syndromes from which to choose a potential diagnosis, paging through a volume, such as Freire-Maia and Pinheiro's corpus, matching their patient's findings to listed syndromes. Medical researchers may want a list of syndromes that share one (monothetic system) or several (polythetic system) traits in order to focus research on a narrowly defined group. Special interest groups may want a list from which they can choose constituencies, and insurance companies and government agencies may want a list to determine for whom to provide (or deny) health-care coverage. Furthermore, various molecular biologists are now promoting classification systems based on gene mutation (e.g., TP63-associated syndromes) or common molecular pathways. The challenge will be to balance comprehensiveness within the classification with usability and accessibility so that the benefits truly serve the needs of researchers, health-care providers, and ultimately the individuals and families directly affected by ectodermal dysplasias. It is also recognized that a new classification approach is an ongoing process and will require periodical reviews or updates. Whatever scheme is developed, however, will have far-reaching application for other groups of disorders for which classification is complicated by the number of interested parties and advances in diagnostic acumen. Consensus among interested parties is necessary for optimizing communication among the diverse groups whether it be for equitable distribution of funds, correctness of diagnosis and treatment, or focusing research efforts.

The leucine-rich amelogenin peptide alters the amelogenin null enamel phenotype.

INTRODUCTION: The amelogenin proteins secreted by ameloblasts during dental enamel development are required for normal enamel structure. Amelx null (KO) mice have hypoplastic, disorganized enamel similar to that of human patients with mutations in the AMELX gene, and provide a model system for studies of the enamel defect amelogenesis imperfecta. Because many amelogenin proteins are present in developing enamel due to RNA alternative splicing and proteolytic processing, understanding the function of individual amelogenins has been challenging. PURPOSE: Our objective was to better understand the role of LRAP, a 59 amino acid leucine-rich amelogenin peptide, in the development of enamel. APPROACH: Teeth from transgenic mice that express LRAP under control of the Amelx regulatory regions were analyzed for mechanical properties, and transgenic males were mated with female KO mice. Male offspring with a null background that were transgene positive or transgene negative were compared to determine phenotypic differences using microcomputed tomography (microCT) and scanning electron microscopy (SEM). RESULTS: Nanoindentation revealed no differences between LRAP transgenic and wild-type murine enamel. Using microCT, LRAPKO enamel volume and density measurements were similar to those from KO mice. However, in etched samples examined by SEM, the organization of the enamel rod pattern was altered by the presence of the LRAP transgene. CONCLUSIONS: The presence of LRAP leads to changes in enamel appearance compared to enamel from KO mice. Expression of a combination of amelogenin transgenes in KO mice may lead to rescue of the individual characteristics of normal enamel.

Human and mouse enamel phenotypes resulting from mutation or altered expression of AMEL, ENAM, MMP20 and KLK4.

Amelogenesis imperfecta (AI) is caused by AMEL, ENAM, MMP20 and KLK4 gene mutations. Mice lacking expression of the AmelX, Enam and Mmp20 genes have been generated. These mouse models provide tools for understanding enamel formation and AI pathogenesis. This study describes the AI phenotypes and relates them to their mouse model counterparts. Human AI phenotypes were determined in a clinical population of AI families and published cases. Human and murine teeth were evaluated using light and electron microscopy. A total of 463 individuals from 54 families were evaluated and mutations in the AMEL, ENAM and KLK4 genes were identified. The majority of human mutations for genes coding enamel nonproteinase proteins (AMEL and ENAM) resulted in variable hypoplasia ranging from local pitting to a marked, generalized enamel thinning. Specific AMEL mutations were associated with abnormal mineralization and maturation defects. Amel and Enam null murine models displayed marked enamel hypoplasia and a complete loss of prism structure. Human mutations in genes coding for the enamel proteinases (MMP20 and KLK4) cause variable degrees of hypomineralization. The murine Mmp20 null mouse exhibits both hypoplastic and hypomineralized defects. The currently available Amel and Enam mouse models for AI exhibit enamel phenotypes (hypoplastic) that are generally similar to those seen in humans. Mmp20 null mice have a greater degree of hypoplasia than humans with MMP20 mutations. Mice lacking expression of the currently known genes associated with the human AI conditions provide useful models for understanding the pathogenesis of these conditions.

A comprehensive analysis of normal variation and disease-causing mutations in the human DSPP gene.

Within nine dentin dysplasia (DD) (type II) and dentinogenesis imperfecta (type II and III) patient/families, seven have 1 of 4 net -1 deletions within the approximately 2-kb coding repeat domain of the DSPP gene while the remaining two patients have splice-site mutations. All frameshift mutations are predicted to change the highly soluble DSPP protein into proteins with long hydrophobic amino acid repeats that could interfere with processing of normal DSPP and/or other secreted matrix proteins. We propose that all previously reported missense, nonsense, and splice-site DSPP mutations (all associated with exons 2 and 3) result in dominant phenotypes due to disruption of signal peptide-processing and/or related biochemical events that also result in interference with protein processing. This would bring the currently known dominant forms of the human disease phenotype in agreement with the normal phenotype of the heterozygous null Dspp (-/+) mice. A study of 188 normal human chromosomes revealed a hypervariable DSPP repeat domain with extraordinary rates of change including 20 slip-replication indel events and 37 predominantly C-to-T transition SNPs. The most frequent transition in the primordial 9-basepair (bp) DNA repeat was a sense-strand CpG site while a CpNpG (CAG) transition was the second most frequent SNP. Bisulfite-sequencing of genomic DNA showed that the DSPP repeat can be methylated at both motifs. This suggests that, like plants and some animals, humans methylate some CpNpG sequences. Analysis of 37 haplotypes of the highly variable DSPP gene from geographically diverse people suggests it may be a useful autosomal marker in human migration studies.

DLX3 c.561_562delCT mutation causes attenuated phenotype of tricho-dento-osseous syndrome.

The distal-less homeobox gene DLX3 is expressed in a variety of tissues including placenta, skin, hair, teeth, and bone. Mutation of DLX3 (c.571_574delGGGG) causes the tricho-dento-osseous syndrome (TDO), characterized by abnormal hair, teeth, and bone. Evaluation of a kindred segregating the DLX3 c.561_562delCT mutation revealed distinct changes in the hair, teeth, and bones as has been observed with the DLX3 c.571_574delGGGG mutation. Previously, the DLX3 c.561_562delCT mutation was associated with autosomal dominant amelogenesis imperfecta with taurodontism. The present study shows that the DLX3 c.560_561delCT mutation causes an attenuated TDO phenotype with less severe hair, tooth, and bone manifestations compared with individuals having the DLX3 c.571_574delGGGG mutation. Careful phenotyping of individuals with allelic DLX3 mutations reveals marked differences in phenotypic severity indicating that the carboxy-terminus of the DLX3 protein is critical in determining its function during development in these different tissues.

Appropriate Fluoride Toothpaste Application: Improving Caregiver Compliance.

Purpose: Fluoridated toothpaste ingestion is associated with increased dental fluorosis prevalence. Current guidelines recommend a smear of fluoridated toothpaste for children younger than three years old and a pea-sized amount for children age three years or older. The purpose of this study was to evaluate two educational approaches to improve caregiver toothpaste dosing. Methods: Eighty-five caregivers of one- to six-year-olds were surveyed and applied toothpaste to a toothbrush as they normally would for their child. All caregivers then received visual aid (VA) counseling on toothpaste dosing (smear equals 0.09 g, with 0.10 mg fluoride and pea equals 0.22 g with 0.25 mg fluoride). Forty-three caregivers were randomly assigned for teach-back (TB) counseling. Each toothpaste application was weighed. Results: Caregivers (N) equals 56) not familiar with toothpaste guidelines applied more toothpaste at baseline than caregivers familiar with the guidelines (P=0.02). The TB group dispensed toothpaste amounts closer to the ideal compared with the VA group. For all caregivers, VA improved dispensing ability (P=0.008). While TB improved dispensing ability, the difference in average deviation from ideal was not statistically significant (P=0.40) between the TB and VA groups. Conclusions: Teach-back and visual aid counseling methods help caregivers dispense more appropriate toothpaste quantities. Dentists should counsel caregivers of children six years old or younger on fluoride toothpaste dosing and use VA and TB to verify dispensing skills.

Diagnosing Developmental Defects of Enamel: Pilot Study of Online Training and Accuracy.

PURPOSE: The purpose of this study was to assess dentists' ability to correctly identify and classify development defects of enamel (DDE). METHODS: The modified DDE (MDDE) index was used to classify enamel defects into two types: (1) enamel hypoplasia-pitted, grooved, or missing enamel; or (2) enamel opacity-translucency of enamel not caused by dental caries or fluorosis (can be either demarcated or diffuse). A panel of six experts selected and scored 36 images using the MDDE, and the consensus score was used as the gold standard score in the evaluation of survey respondents. A short training table was developed to match training images to descriptors for the MDDE. A survey, including the training table, was then distributed electronically to 2,036 U.S. dentists and expanded function dental assistants from the Indian Health Service and 6,174 members of American Academy of Pediatric Dentistry. The percent of correct responses was evaluated for each image. RESULTS: Survey respondents (348 total) showed great variability in correct responses for each image, ranging from 41 to 97 percent, for each category of the MDDE. CONCLUSIONS: Enhanced training and calibration on the ability of dental providers is needed to identify the different types of development defects of enamel.

Cloning of rat amelotin and localization of the protein to the basal lamina of maturation stage ameloblasts and junctional epithelium.

Formation of tooth enamel is a very complex process in which a specific set of proteins secreted by ameloblasts play a primordial role. As part of a screening procedure to identify novel proteins secreted by EO (enamel organ) cells of rat incisors, we isolated a partial cDNA fragment (EO-017) that is the homologue of the recently described mouse Amtn (amelotin) gene [Iwasaki, Bajenova, Somogyi-Ganss, Miller, Nguyen, Nourkeyhani, Gao, Wendel and Ganss (2005) J. Dent. Res. 84, 1127-1132]. Presented herein is the cloning of rat and pig full-length cDNAs with their deduced protein sequences. Detailed expression profiling by Northern-blot analysis and RT (reverse transcriptase)-PCR on rat and mouse tissues revealed highest expression in the mandible, more specifically in the maturation stage of the EO. Among all tissues tested, low expression was detected only in periodontal ligament, lung, thymus and gingiva. In silico analyses revealed that the Amtn gene is highly conserved in seven other mammals, but is absent from fish, birds and amphibians. The Amtn protein is enriched in proline, leucine, glutamine and threonine (52% of total) and contains a perfectly conserved protein kinase CK2 phosphorylation site. Transient transfection experiments in HEK-293 cells (human embryonic kidney cells) showed that secreted Amtn is post-translationally modified possibly through O-linked oligosaccharides on threonine residues. In concordance with its predominant expression site, immunofluorescence localization within the rat and mouse mandibles revealed Amtn localized to the basal lamina of maturation stage ameloblasts of incisors and unerupted molars. Intense Amtn protein expression was also detected in the internal basal lamina of junctional epithelium in molars. The peculiar and unique cellular localization of Amtn suggests a role in cell adhesion.

A systematic review of the association between consumption of sugar-containing beverages and excess weight gain among children under age 12.

OBJECTIVE: A systematic review was conducted to address this clinical question: Does consumption of (non-dairy) sugar-containing beverages (SCBs) among children under age 12 result in excess weight gain? METHODS: The authors searched four databases for controlled trials (randomized and non-randomized) and cohort studies published in English through March 29, 2016: PubMed, EMBASE, Cochrane Database of Systematic Reviews, CINAHL. Initial and full-text screening, data abstraction, and risk of bias assessment were performed independently and in duplicate. RESULTS: Thirty-eight studies met inclusion criteria for this systematic review. One was a randomized controlled trial, and 37 were cohort studies. Though the results of these studies were mixed, the majority demonstrated a statistically significant positive association between SCB consumption in children under age 12 and total adiposity and central adiposity. In contrast, most studies that assessed 100 percent fruit juice consumption only with either total adiposity or central adiposity did not support an association. Among only children under age 5 at baseline, no studies examined central adiposity, but nearly all studies examining SCBs and total adiposity, and a majority examining only fruit juice consumption, demonstrated a statistically significant positive association. CONCLUSION: Our results support a statistically significant positive association between SCBs and total and central adiposity among children under age 12. This association is most consistent for total adiposity among children <5. Our results for 100 percent fruit juice only suggest differences by age, as most studies among those < 12 were negative but most among those <5 were positive.

Meeting report: a hard look at the state of enamel research.

The Encouraging Novel Amelogenesis Models and Ex vivo cell Lines (ENAMEL) Development workshop was held on 23 June 2017 at the Bethesda headquarters of the National Institute of Dental and Craniofacial Research (NIDCR). Discussion topics included model organisms, stem cells/cell lines, and tissues/3D cell culture/organoids. Scientists from a number of disciplines, representing institutions from across the United States, gathered to discuss advances in our understanding of enamel, as well as future directions for the field.