Single nucleotide polymorphisms of enamel formation genes and early childhood caries - systematic review, gene-based, gene cluster and meta-analysis.

Introduction: Genetic polymorphisms of genes regulating amelogenesis can alter susceptibility to Early Childhood Caries (ECC). This systematic review aims to analyze associations between single-nucleotide polymorphisms of enamel formation genes and ECC. Methods: Search was conducted across PUBMED, CINAHL, LILACS, SCOPUS, EMBASE, Web of Science, Genome-Wide Association Studies databases from January 2003 to September 2022. This was supplemented by hand search. Totally 7124 articles were identified and 21 articles that satisfied the inclusion criteria proceeded to data extraction. Quality assessment was done using the Q-Genie tool. Results: Quantitative synthesis revealed that homozygous genotype AA of rs12640848 was significantly higher in children with ECC with an odds ratio of 2.36. Gene-based analysis revealed significant association between six variants of AMBN, four variants of KLK4, two variants of MMP20, and a single variant of each of MMP9 and MMP13 genes and ECC. The Bonferroni corrected-log10 P value of amelogenesis gene Cluster was 2.25 (0.05/88 = 5.6 × 10-4). Search Tool for Retrieval of Interacting Genes and Proteins plot constructed to comprehend the protein-protein interaction revealed the presence of four functional clusters. Gene function prediction using Multiple Association Network Integration Algorithm revealed that physical interaction between these genes was 69.3%. Conclusion: Polymorphisms of genes regulating amelogenesis can influence the susceptibility to ECC. AA genotype of rs12640848 may increase the susceptibility to ECC. Gene-based analysis revealed a significant association between multiple polymorphisms of genes regulating amelogenesis and ECC.

Autophagy Plays a Crucial Role in Ameloblast Differentiation.

Tooth enamel is generated by ameloblasts. Any failure in amelogenesis results in defects in the enamel, a condition known as amelogenesis imperfecta. Here, we report that mice with deficient autophagy in epithelial-derived tissues (K14-Cre;Atg7F/F and K14-Cre;Atg3F/F conditional knockout mice) exhibit amelogenesis imperfecta. Micro-computed tomography imaging confirmed that enamel density and thickness were significantly reduced in the teeth of these mice. At the molecular level, ameloblast differentiation was compromised through ectopic accumulation and activation of NRF2, a specific substrate of autophagy. Through bioinformatic analyses, we identified Bcl11b, Dlx3, Klk4, Ltbp3, Nectin1, and Pax9 as candidate genes related to amelogenesis imperfecta and the NRF2-mediated pathway. To investigate the effects of the ectopic NRF2 pathway activation caused by the autophagy deficiency, we analyzed target gene expression and NRF2 binding to the promoter region of candidate target genes and found suppressed gene expression of Bcl11b, Dlx3, Klk4, and Nectin1 but not of Ltbp3 and Pax9. Taken together, our findings indicate that autophagy plays a crucial role in ameloblast differentiation and that its failure results in amelogenesis imperfecta through ectopic NRF2 activation.

Ift88 regulates enamel formation via involving Shh signaling.

OBJECTIVES: The ciliopathies are a wide spectrum of human diseases, which are caused by perturbations in the function of primary cilia. Tooth enamel anomalies are often seen in ciliopathy patients; however, the role of primary cilia in enamel formation remains unclear. MATERIALS AND METHODS: We examined mice with epithelial conditional deletion of the ciliary protein, Ift88, (Ift88fl / fl ;K14Cre). RESULTS: Ift88fl / fl ;K14Cre mice showed premature abrasion in molars. A pattern of enamel rods which is determined at secretory stage, was disorganized in Ift88 mutant molars. Many amelogenesis-related molecules expressing at the secretory stage, including amelogenin and ameloblastin, enamelin, showed significant downregulation in Ift88 mutant molar tooth germs. Shh signaling is essential for amelogenesis, which was found to be downregulated in Ift88 mutant molar at the secretory stage. Application of Shh signaling agonist at the secretory stage partially rescued enamel anomalies in Ift88 mutant mice. CONCLUSION: Findings in the present study indicate that the function of the primary cilia via Ift88 is critical for the secretory stage of amelogenesis through involving Shh signaling.

Overexpression of ameloblastin in secretory ameloblasts results in demarcated, hypomineralized opacities in enamel.

Introduction: Developmental defects of the enamel manifest before tooth eruption and include amelogenesis imperfecta, a rare disease of underlying gene mutations, and molar-incisor hypomineralization (MIH), a prevalent disease in children originating from environmental and epigenetic factors. MIH enamel presents as the abnormal enamel marked by loss of translucency, demarcation between the healthy and affected enamel, and reduced mineral content. The pathophysiology of opaque, demarcated enamel lesions is not understood; however, the retention of enamel proteins in the matrix has been suggested. Ameloblastin (Ambn) is an enamel protein of the secreted calcium-binding phosphoproteins (SCPPs) critical for enamel formation. When the Ambn gene is mutated or deleted, teeth are affected by hypoplastic amelogenesis imperfecta. Methods: In this study, enamel formation in mice was analyzed when transgenic Ambn was overexpressed from the amelogenin promoter encoding full-length Ambn. Ambn was under- and overexpressed at six increasing concentrations in separate mouse lines. Results: Mice overexpressing Ambn displayed opaque enamel at low concentrations and demarcated lesions at high concentrations. The severity of enamel lesions increased starting from the inner enamel close to the dentino-enamel junction (DEJ) to span the entire width of the enamel layer in demarcated areas. Associated with the opaque enamel were 17-kDa Ambn cleavage products, a prolonged secretory stage, and a thin basement membrane in the maturation stage. Ambn accumulations found in the innermost enamel close to the DEJ and the mineralization front correlated with reduced mineral content. Demarcated enamel lesions were associated with Ambn species of 17 kDa and higher, prolonged secretory and transition stages, a thin basement membrane, and shortened maturation stages. Hypomineralized opacities were delineated against the surrounding mineralized enamel and adjacent to ameloblasts detached from the enamel surface. Inefficient Ambn cleavage, loss of contact between ameloblasts, and the altered basement membrane curtailed the endocytic activity; thus, enamel proteins remained unresorbed in the matrix. Ameloblasts have the ability to distinguish between Ambn concentration and Ambn cleavage products through finely tuned feedback mechanisms. The under- or overexpression of Ambn in murine secretory ameloblasts results in either hypoplastic amelogenesis imperfecta or hypomineralization with opaque or sharply demarcated boundaries of lesions, similar to MIH.

Response of TGF-ß isoforms in epithelial-mesenchymal transition of enamel epithelial cells.

OBJECTIVE: During enamel formation, transforming growth factor-beta (TGF-ß) isoforms exhibit different activities for gene expression, apoptosis, and endocytosis. This study aimed to investigate the differential response of TGF-ß isoforms to epithelial-mesenchymal transition (EMT) in enamel epithelial cells. DESIGN: Using a mouse enamel epithelial cell line (mHAT9d) cultured in the presence of each TGF-ß isoform, (1) the morphological changes in EMT were explored, (2) EMT-related genes were analyzed by next-generation sequencing (NGS), (3) TGF-ß pathway for EMT was identified by inhibition experiments, and (4) the expression of the TGF-ß receptor gene in response to the binding affinity of the TGF-ß isoform were analyzed. RESULTS: EMT was observed in mHAT9d cultured in the presence of TGF-ß1 and ß3 but not TGF-ß2. The expression of both epithelial and mesenchymal marker genes was observed in mHAT9d exhibiting EMT. NGS analysis suggested extracellular signal-regulated kinase (ERK) and Rho pathways as TGF-ß signaling pathways associated with EMT. However, EMT in mHAT9d cultured in the presence of TGF-ß1 or ß3 occurred even in presence of an ERK1/2 inhibitor and was suppressed by Rho-kinase inhibitor. The expression of co-receptors for TGF-ß signaling in mHAT9d cells reduced following stimulation with each TGF-ß isoform. In contrast, endoglin levels increased following TGF-ß1 or ß3 stimulation, but no change was noted in response to TGF-ß2. CONCLUSIONS: We propose that in TGF-ß-stimulated enamel epithelial cells, EMT mainly occurred via the Rho signaling pathway, and the differences in response across TGF-ß isoforms were due to their endoglin-mediated binding affinity for the TGF-ß receptor.

Focusing on Hippo Pathway in Stem Cells of Oral Origin, Enamel Formation and Periodontium Regeneration.

Hippo pathway is a cellular regulatory pathway composed of core molecules such as MST1/2, LATS1/2, SAV1, MOB1A/B and downstream YAP/TAZ. Fully involved in regulating cell proliferation, differentiation, migration and apoptosis, the Hippo pathway is critical in regulating stem cells of oral origin, for instance, DPSCs and PDLSCs, enamel formation and periodontium regeneration. Here, we summarized the Hippo pathway involved in these progresses and concluded crosstalks of the Hippo pathway with BCL-2, ERK1/2, ROCK, TGF-ß/BMP and Wnt/ß-catenin pathways, hoping to provide foundation for further clinical therapy.

How prenatal environmental factors affect rat molar enamel formation?

Amelogenin (AMELX) and ameloblastin (AMBN) are crucial for enamel formation, and interruptions in the production of these proteins may cause enamel defects. We investigated how prenatal environmental factors (chronic stress, bisphenol A (BPA), amoxicillin, and lipopolysaccharide (LPS)) affect AMELX and AMBN production of ameloblasts. Fifteen pregnant Sprague-Dawley rats were divided into four experimental groups and a control group. Chronic-stress group rats were exposed to a 12:12 light/light cycle (LL) from day E18 until delivery. BPA group rats were orally administered 5 µg/kg BPA daily from day E1 until delivery. Amoxicillin group rats were injected 100 mg/kg amoxicillin daily from day E18 until delivery. LPS-infection group rats were injected 125 µg/kg bacterial LPS once on day E18. Seven pups from the control group and ten pups from the experimental groups were euthanized on P10. Sections were stained with hematoxylin and eosin (H&E) and Gomori's one-step trichrome staining (GT) and incubated with rabbit polyclonal antibodies to AMELX and AMBN, to evaluate staining intensity at ameloblast stages. The surface morphology was evaluated with a stereomicroscope. AMELX (p = 0.008, p = 0.0001, p = 0.009) and AMBN (p = 0.002, p = 0.001, p = 0.0001) staining of all groups were significantly lower than that of the control group in the secretory, transitional, and maturation stages. Abnormal enamel matrix formation was observed in the H&E and GT staining sections of all experimental groups. Yellowish coloration of the amoxicillin group was observed in morphologic evaluation.

von Willebrand factor D and EGF domains regulate ameloblast differentiation and enamel formation.

Cell- and tissue-specific extracellular matrix (ECM) composition plays an important role in organ development, including teeth, by regulating cell behaviors, such as cell proliferation and differentiation. Here, we demonstrate for the first time that von Willebrand factor D and epidermal growth factor (EGF) domains (Vwde), a previously uncharacterized ECM protein, is specifically expressed in teeth and regulates cell proliferation and differentiation in inner enamel epithelial cells (IEEs) and enamel formation. We identified the Vwde as a novel ECM protein through bioinformatics using the NCBI expressed sequence tag database for mice. Vwde complementary DNA encodes 1773 amino acids containing a signal peptide, a von Willebrand factor type D domain, and tandem calcium-binding EGF-like domains. Real-time polymerase chain reaction demonstrated that Vwde is highly expressed in tooth tissue but not in other tissues including the brain, lung, heart, liver, kidney, and bone. In situ hybridization revealed that the IEEs expressed Vwde messenger RNA in developing teeth. Immunostaining showed that VWDE was localized at the proximal and the distal ends of the pericellular regions of the IEEs. Vwde was induced during the differentiation of mouse dental epithelium-derived M3H1 cells. Vwde-transfected M3H1 cells secreted VWDE protein into the culture medium and inhibited cell proliferation, whereas ameloblastic differentiation was promoted. Furthermore, Vwde increased the phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B and strongly induced the expression of the intercellular junction protein, N-cadherin (Ncad). Interestingly, the suppression of endogenous Vwde inhibited the expression of Ncad. Finally, we created Vwde-knockout mice using the CRISPR-Cas9 system. Vwde-null mice showed low mineral density, rough surface, and cracks in the enamel, indicating the enamel hypoplasia phenotype. Our findings suggest that Vwde assembling the matrix underneath the IEEs is essential for Ncad expression and enamel formation.

Enamel daily secretion rates of deciduous molars from a global sample of children.

OBJECTIVE: To investigate and describe the variation in enamel daily secretion rates (DSRs) of naturally exfoliated deciduous molars (n = 345) from five modern-day populations (Aotearoa New Zealand, Britain, Canada, France, and Sweden). DESIGN: Each tooth was thin sectioned and examined using a high-powered Olympus BX51 microscope and DP25 digital microscope camera. Mean DSRs were recorded for the inner, mid, and outer regions of cuspal and lateral enamel, excluding enamel nearest the enamel-dentin junction and at the outermost crown surface. RESULTS: Mean DSRs did not vary significantly between populations, or by sex. Cuspal enamel grew slightly faster than lateral enamel (mean difference 0.16 µm per day; p < 0.001). The trajectory of DSRs remained relatively constant from inner to outer cuspal enamel and increased slightly in lateral enamel (p = 0.003). CONCLUSIONS: The DSRs of deciduous molars from modern-day children are remarkably consistent when compared among populations. While growth rates are faster in cuspal than lateral enamel, the trajectory of enamel formation changes only slightly from inner to outer regions. The trajectory of DSRs for deciduous molars differs to that of permanent molar enamel, which typically display a steep increase in matrix deposition from inner to outer enamel.

Overexpression of miR-1306-5p, miR-3195, and miR-3914 Inhibits Ameloblast Differentiation through Suppression of Genes Associated with Human Amelogenesis Imperfecta.

Amelogenesis imperfecta is a congenital form of enamel hypoplasia. Although a number of genetic mutations have been reported in humans, the regulatory network of these genes remains mostly unclear. To identify signatures of biological pathways in amelogenesis imperfecta, we conducted bioinformatic analyses on genes associated with the condition in humans. Through an extensive search of the main biomedical databases, we found 56 genes in which mutations and/or association/linkage were reported in individuals with amelogenesis imperfecta. These candidate genes were further grouped by function, pathway, protein-protein interaction, and tissue-specific expression patterns using various bioinformatic tools. The bioinformatic analyses highlighted a group of genes essential for extracellular matrix formation. Furthermore, advanced bioinformatic analyses for microRNAs (miRNAs), which are short non-coding RNAs that suppress target genes at the post-transcriptional level, predicted 37 candidates that may be involved in amelogenesis imperfecta. To validate the miRNA-gene regulation association, we analyzed the target gene expression of the top seven candidate miRNAs: miR-3195, miR-382-5p, miR-1306-5p, miR-4683, miR-6716-3p, miR-3914, and miR-3935. Among them, miR-1306-5p, miR-3195, and miR-3914 were confirmed to regulate ameloblast differentiation through the regulation of genes associated with amelogenesis imperfecta in AM-1 cells, a human ameloblastoma cell line. Taken together, our study suggests a potential role for miRNAs in amelogenesis imperfecta.

An inconstant biorhythm: The changing pace of Retzius periodicity in human permanent teeth.

OBJECTIVES: Human tooth enamel retains evidence of growth in the form of Retzius lines. The number of daily growth increments between the regularly occurring lines defines their repeat interval, or periodicity. Retzius periodicity is often incorporated into enamel formation times, age-at-death reconstructions, or used to provide a basis from which to explore an underlying biorhythm. Biological anthropologists typically assume that RP remains constant within an individual and does not vary along the tooth-row. Here, we test that assumption. MATERIALS AND METHODS: RP was calculated from n = 223 thin sections of human permanent teeth from individuals of British and southern African origin. Forty individuals provided multiple teeth (n = 102 teeth) and a further 121 individuals each provided a single tooth. RESULTS: We report first evidence that RP of permanent teeth does not always remain constant within an individual. Of those individuals that provided multiple teeth, 42% (n = 17/40) demonstrated a decrease in RP along the tooth row, with most shifting by two or more days (n = 11). Across the entire sample, mean RP of anterior teeth was significantly higher than molars. Mean premolar RP tended to be intermediate between anterior teeth and molars. DISCUSSION: Our data do not support the assumption that RP invariably remains constant within the permanent teeth of an individual. Transferring RP from molars to incisors within an individual can result in a miscalculation of formation time and age-at-death by up to 1 year. Implications for biological anthropologists and the source of the underlying long period biorhythm are discussed.

Characteristics of the transverse 2D uniserial arrangement of rows of decussating enamel rods in the inner enamel layer of mouse mandibular incisors.

The 2D arrangement of rows of enamel rods with alternating (decussating) tilt angles across the thickness of the inner layer in rat and mouse incisor enamel is well known and assumed to occur in a uniform and repetitive pattern. Some irregularities in the arrangement of rows have been reported, but no detailed investigation of row structure across the entire inner enamel layer currently exists. This investigation was undertaken to determine if the global row pattern in mouse mandibular incisor enamel is predominately regular in nature with only occasional anomalies or if rows of enamel rods have more spatial complexity than previously suspected. The data from this investigation indicate that rows of enamel rods are highly variable in length and have complex transverse arrangements across the width and thickness of the inner enamel layer. The majority of rows are short or medium in length, with 87% having < 100 rods per row. The remaining 13% are long rows (with 100-233 rods per row) that contain 46% of all enamel rods seen in transverse sections. Variable numbers of rows were associated with the lateral, central and mesial regions of the enamel layer. Each region contained different ratios of short, medium and long rows. A variety of relationships was found along the transverse length of rows in each region, including uniform associations of alternating rod tilts between neighboring rows, and instances where two rows having the same rod tilt were paired for variable distances then moved apart to accommodate rows of opposite tilt. Sometimes a row appeared to branch into two rows with the same tilt, or conversely where two rows merged into one row depending upon the mesial-to-lateral direction in which the row was viewed. Some rows showed both pairing and branching/merging along their length. These tended to be among the longest rows identified, and they often crossed the central region with extensions into the lateral and mesial regions. The most frequent row arrangement was a row of petite length nestled at the side of another row having the same rod tilt (30% of all rows). These were termed 'focal stacks' and may relate to the evolution of uniserial rat and mouse incisor enamel from a multilayered ancestor. The mesial and lateral endpoints of rows also showed complex arrangements with the dentinoenamel junction (DEJ), the inner enamel layer itself, and the boundary area to the outer enamel layer. It was concluded that the diversity in row lengths and various spatial arrangements both within and between rows across the transverse plane provides a method to interlock the enamel layer across each region and keep the enamel layer compact relative to the curving DEJ surface. The uniserial pattern for rows in mouse mandibular incisors is not uniform, but diverse and very complex.

AMBN mutations causing hypoplastic amelogenesis imperfecta and Ambn knockout-NLS-lacZ knockin mice exhibiting failed amelogenesis and Ambn tissue-specificity.

BACKGROUND: Ameloblastin (AMBN) is a secreted matrix protein that is critical for the formation of dental enamel and is enamel-specific with respect to its essential functions. Biallelic AMBN defects cause non-syndromic autosomal recessive amelogenesis imperfecta. Homozygous Ambn mutant mice expressing an internally truncated AMBN protein deposit only a soft mineral crust on the surface of dentin. METHODS: We characterized a family with hypoplastic amelogenesis imperfecta caused by AMBN compound heterozygous mutations (c.1061T>C; p.Leu354Pro/ c.1340C>T; p.Pro447Leu). We generated and characterized Ambn knockout/NLS-lacZ (AmbnlacZ/lacZ ) knockin mice. RESULTS: No AMBN protein was detected using immunohistochemistry in null mice. ß-galactosidase activity was specific for ameloblasts in incisors and molars, and islands of cells along developing molar roots. AmbnlacZ/lacZ 7-week incisors and unerupted (D14) first molars showed extreme enamel surface roughness. No abnormalities were observed in dentin mineralization or in nondental tissues. Ameloblasts in the AmbnlacZ/lacZ mice were unable to initiate appositional growth and started to degenerate and deposit ectopic mineral. No layer of initial enamel ribbons formed in the AmbnlacZ/lacZ mice, but pockets of amelogenin accumulated on the dentin surface along the ameloblast distal membrane and within the enamel organ epithelia (EOE). NLS-lacZ signal was positive in the epididymis and nasal epithelium, but negative in ovary, oviduct, uterus, prostate, seminal vesicles, testis, submandibular salivary gland, kidney, liver, bladder, and bone, even after 15 hr of incubation with X-gal. CONCLUSIONS: Ameloblastin is critical for the initiation of enamel ribbon formation, and its absence results in pathological mineralization within the enamel organ epithelia.

A Review of the Role of Amelogenin Protein in Enamel Formation and Novel Experimental Techniques to Study its Function.

Amelognein protein plays a vital role in the formation and mineralization of enamel matrix. Amelogenin structure is complex in nature and researchers have studied it with different experimental techniques. Considering its important role, there is a need to understand this important protein, which has been discussed in detail in this review. In addition, various experimental techniques to study amelogenin protein used previously have been tackled along with their advantages and disadvantages. A selection of 67 relevant articles/book chapters was included in this study. The review concluded that amelogenins act as nanospheres or spacers for the growth of enamel crystals. Various experimental techniques can be used to study amelogenins, however, their advantages and drawbacks should be kept in mind before performing analysis.

Quantitative analysis of the core 2D arrangement and distribution of enamel rods in cross-sections of mandibular mouse incisors.

Considerable descriptive information about the overall organization of mouse mandibular incisor enamel is available but almost nothing is known about the quantitative characteristics of enamel rod arrangement and distribution in these teeth. This has important implications concerning cell movement during the secretory stage because each ameloblast makes one enamel rod. Knowing how many enamel rods are cut open in a cross-section of the enamel layer could provide insights into understanding the dynamics of how groups of ameloblasts form the enamel layer. In this study, cross-sections of fully mineralized enamel were cut on 24 mandibular mouse incisors, polished and etched, and imaged by scanning electron microscopy in backscatter mode. Montaged maps of the entire enamel layer were made at high magnification and the enamel rod profiles in each map were color-coded based upon rod category. Quantitative analyses of each color layer in the maps were then performed using standard routines available in imagej. The data indicated that that there were on average 7233 ± 575 enamel rod profiles per cross-section in mandibular incisors of 7-week-old mice, with 70% located in the inner enamel layer, 27% located in the outer enamel layer, and 3% positioned near the mesial and lateral cementoenamel junctions. All enamel rod profiles showed progressive increases in tilt angles, some very large in magnitude, from the lateral to mesial sides of the enamel layer, whereas only minor variations in tilt angle were found relative to enamel thickness at given locations across the enamel layer. The decussation angle between alternating rows of rod profiles within the inner enamel layer was fairly constant from the lateral to central labial sides of the enamel layer, but it increased dramatically in the mesial region of the enamel layer. The packing density of all rod profiles decreased from lateral to central labial regions of the enamel layer and then in progressing mesially, decreased slightly (inner enamel, mesial tilt), increased slightly (outer enamel layer) or almost doubled in magnitude (inner enamel, lateral tilt). It was concluded that these variations in rod tilt angle and packing densities are adaptations that allow the tooth to maintain a sharp incisal edge and shovel-shape as renewing segments formed by around 7200 ameloblasts are brought onto the occluding surface of the tooth by continuous renewal.

Pig enamel revisited - Incremental markings in enamel of wild boars and domestic pigs.

The nature and periodicity of incremental markings in pig enamel is currently debated. To broaden the basis for a correct interpretation of growth marks in pig enamel, we analyzed their periodicity in teeth of wild boars and domestic pigs. For that, the numbers of enamel incremental markings were recorded in ground sections and compared with crown formation times for the respective teeth derived from literature data on tooth development and eruption in Sus scrofa. Our study revealed that laminations with a daily periodicity are the dominant incremental feature of pig enamel. In wild boar M3s, daily enamel secretion (apposition) rates ranged between a minimum of 6.1 µm in the inner and a maximum of 30.6 µm in the outer enamel. Long-period (supra-daily) incremental markings were present as perikymata at the outer enamel surface (OES). Contrary to the situation in primate enamel, in pig enamel the long-period incremental lines terminating in perikyma grooves were mostly structurally indistinguishable from the daily laminations. Typically, five sub-daily increments were present between successive laminations. The incremental pattern in pig enamel can be misinterpreted if the laminations are mistaken for long-period markings (striae of Retzius) and the sub-daily growth marks for daily prism cross-striations. The findings of the present study demonstrate the critical importance of correctly characterizing the incremental markings and their periodicity in enamel, and caution against an uncritical transfer of the interpretation of the nature of incremental markings in primate enamel to other mammalian taxa.

Current Guidance for Fluoride Intake: Is It Appropriate?

The purpose of this report is to examine critically the appropriateness of the current guidance for fluoride intake in the population (0.05-0.07 mg F/kg bodyweight/d), consider whether changes to the current guidance are desirable, and suggest further research that will strengthen the evidence base for future decisions on guidance/advice in this area. The benefits and the risks of using fluoride particularly concern preschool children because it is at this age that excessive fluoride intake may result in dental fluorosis. Data from mostly cross-sectional studies show a wide variation in exposure and a considerable variation in the amount of fluoride ingested. Fluorosis, mostly mild, is commonly observed. For considering changes in current guidance, there is a need for more knowledge on the relationship between exposure to fluoride at an early age and the development of fluorosis. For that, prospective epidemiological studies with sufficiently large and representative samples of children are required. It is also important to study children in communities both with and without water fluoridation and to include populations where salt or milk fluoridation is used. There is also a need for professional agreement on acceptable levels of mild and moderate/severe fluorosis and a more comprehensive knowledge on the appreciation of mild fluorosis among the public.