Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When transplanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.
Mice ; Animals ; Durapatite/metabolism* ; Dental Enamel/metabolism* ; Ameloblasts/metabolism* ; Amelogenesis ; Stem Cells ; Organoids

Tooth enamel is a complex mineralized tissue consisting of long and parallel apatite crystals configured into decussating enamel rods. In recent years, multiple approaches have been introduced to generate or regenerate this highly attractive biomaterial characterized by great mechanical strength paired with relative resilience and tissue compatibility. In the present review, we discuss five pathways toward enamel tissue engineering, (i) enamel synthesis using physico-chemical means, (ii) protein matrix-guided enamel crystal growth, (iii) enamel surface remineralization, (iv) cell-based enamel engineering, and (v) biological enamel regeneration based on de novo induction of tooth morphogenesis. So far, physical synthesis approaches using extreme environmental conditions such as pH, heat and pressure have resulted in the formation of enamel-like crystal assemblies. Biochemical methods relying on enamel proteins as templating matrices have aided the growth of elongated calcium phosphate crystals. To illustrate the validity of this biochemical approach we have successfully grown enamel-like apatite crystals organized into decussating enamel rods using an organic enamel protein matrix. Other studies reviewed here have employed amelogenin-derived peptides or self-assembling dendrimers to re-mineralize mineral-depleted white lesions on tooth surfaces. So far, cell-based enamel tissue engineering has been hampered by the limitations of presently existing ameloblast cell lines. Going forward, these limitations may be overcome by new cell culture technologies. Finally, whole-tooth regeneration through reactivation of the signaling pathways triggered during natural enamel development represents a biological avenue toward faithful enamel regeneration. In the present review we have summarized the state of the art in enamel tissue engineering and provided novel insights into future opportunities to regenerate this arguably most fascinating of all dental tissues.
Acid Etching, Dental ; Amelogenin ; Biomimetics ; trends ; Dental Enamel ; metabolism ; Dental Enamel Proteins ; Dentistry ; trends ; Tissue Engineering ; methods ; Tooth Remineralization

OBJECTIVE: To investigate the regulation mechanism of RhoA signaling pathway during the enamel formation by using the EGFP-RhoA^{Dominant Negative} (EGFP-RhoA^DN) transgenic mice model, from the aspect of adherens junctions, and to provide a theory basis for mechanism of enamel development defects. METHODS: The enamel thickness of mandibular first molars of EGFP-RhoA^DN transgenic mice and wild type (WT) mice were observed by scanning electronic microscopy at 20 kV, and the enamel thickness of the distal face of the central cusp was measured at 10 locations via analysis by ImageJ (Rasband, 1997-2009). The enamel organs from mandibular first molars from postnatal-4-day (P4) EGFP-RhoA^DN mice and wild type mice were isolated, and the total RNA and protein were extracted from the epithelium of the enamel organs. The expression level of the adherens junctions components in ameloblasts layer of the postnatal-4-day EGFP-RhoA^DN transgenic mice and wild type mice mandibular first molars were detected by real-time PCR and Western blot assay. RESULTS: The EGFP-RhoA^DN transgenic mice had decreased enamel thickness in their bilateral mandibular first molars versus those of control group (n=20), and enamel thickness was (84.60±0.20) μm vs. (106.24±0.24) μm, P<0.05. The protein expressions of E-cadherin, α-E-catenin and pan-cadherin in ameloblasts layer of postnatal-4-day EGFP-RhoA^DN transgenic mice molars were down-regulated, and the protein level of β-catenin in ameloblasts layer of P4 EGFP-RhoA^DN transgenic mice molars was up-regulated. The mRNA level of E-cadherin in ameloblasts layer of P4 EGFP-RhoA^DN transgenic mice molars was down-regulated versus that of WT mice, and the gene expression of E-cadherin was 0.93±0.01 vs. 1.00±0.02, P<0.05. The mRNA level of β-catenin in ameloblasts layer of P4 EGFP-RhoA^DN transgenic mice molars was up-regulated versus that of WT mice, and the gene expression of β-catenin was 1.23±0.03 vs. 1.00±0.05, P<0.05. CONCLUSION In the mandibular first molars of EGFP-RhoA^DN transgenic mice, the enamel formation was disrupted and the adherens junctions of EGFP-RhoA^DN transgenic mice ameloblasts were implicated during amelogenesis. RhoA signaling pathway may play a critical role in enamel development by altering the adherens junctions in ameloblasts.
Adherens Junctions ; Ameloblasts ; Amelogenesis ; Animals ; Antigens, CD ; Cadherins/metabolism* ; Dental Enamel/metabolism* ; Enamel Organ ; Humans ; Mice ; Mice, Transgenic ; Molar ; Signal Transduction ; alpha Catenin ; beta Catenin ; rhoA GTP-Binding Protein/physiology*

Histone methylation is one of the most widely studied post-transcriptional modifications. It is thought to be an important epigenetic event that is closely associated with cell fate determination and differentiation. To explore the spatiotemporal expression of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) epigenetic marks and methylation or demethylation transferases in tooth organ development, we measured the expression of SET7, EZH2, KDM5B and JMJD3 via immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis in the first molar of BALB/c mice embryos at E13.5, E15.5, E17.5, P0 and P3, respectively. We also measured the expression of H3K4me3 and H3K27me3 with immunofluorescence staining. During murine tooth germ development, methylation or demethylation transferases were expressed in a spatial-temporal manner. The bivalent modification characterized by H3K4me3 and H3K27me3 can be found during the tooth germ development, as shown by immunofluorescence. The expression of SET7, EZH2 as methylation transferases and KDM5B and JMJD3 as demethylation transferases indicated accordingly with the expression of H3K4me3 and H3K27me3 respectively to some extent. The bivalent histone may play a critical role in tooth organ development via the regulation of cell differentiation.
Animals ; Cell Differentiation ; physiology ; DNA-Binding Proteins ; analysis ; Dental Papilla ; embryology ; Embryo, Mammalian ; Enamel Organ ; embryology ; Enhancer of Zeste Homolog 2 Protein ; Epigenesis, Genetic ; physiology ; Gene Expression Regulation, Developmental ; Histone-Lysine N-Methyltransferase ; analysis ; Histones ; metabolism ; Jumonji Domain-Containing Histone Demethylases ; analysis ; Lysine ; metabolism ; Methylation ; Mice ; Mice, Inbred BALB C ; Odontogenesis ; physiology ; Polycomb Repressive Complex 2 ; analysis ; Protein Processing, Post-Translational ; physiology ; Tooth Germ ; embryology

The objective of the study was to analyse Streptococcus mutans biofilms grown under different dietary conditions by using multifaceted methodological approaches to gain deeper insight into the cariogenic impact of carbohydrates. S. mutans biofilms were generated during a period of 24 h in the following media: Schaedler broth as a control medium containing endogenous glucose, Schaedler broth with an additional 5% sucrose, and Schaedler broth supplemented with 1% xylitol. The confocal laser scanning microscopy (CLSM)-based analyses of the microbial vitality, respiratory activity (5-cyano-2,3-ditolyl tetrazolium chloride, CTC) and production of extracellular polysaccharides (EPS) were performed separately in the inner, middle and outer biofilm layers. In addition to the microbiological sample testing, the glucose/sucrose consumption of the biofilm bacteria was quantified, and the expression of glucosyltransferases and other biofilm-associated genes was investigated. Xylitol exposure did not inhibit the viability of S. mutans biofilms, as monitored by the following experimental parameters: culture growth, vitality, CTC activity and EPS production. However, xylitol exposure caused a difference in gene expression compared to the control. GtfC was upregulated only in the presence of xylitol. Under xylitol exposure, gtfB was upregulated by a factor of 6, while under sucrose exposure, it was upregulated by a factor of three. Compared with glucose and xylitol, sucrose increased cell vitality in all biofilm layers. In all nutrient media, the intrinsic glucose was almost completely consumed by the cells of the S. mutans biofilm within 24 h. After 24 h of biofilm formation, the multiparametric measurements showed that xylitol in the presence of glucose caused predominantly genotypic differences but did not induce metabolic differences compared to the control. Thus, the availability of dietary carbohydrates in either a pure or combined form seems to affect the cariogenic potential of S. mutans biofilms.
Bacterial Load ; drug effects ; Bacteriological Techniques ; Biofilms ; drug effects ; Cariogenic Agents ; metabolism ; pharmacology ; Culture Media ; Dental Enamel ; microbiology ; Fluorescent Dyes ; Gene Expression Regulation, Bacterial ; drug effects ; Gene Expression Regulation, Enzymologic ; drug effects ; Genotype ; Glucose ; metabolism ; Glucosyltransferases ; metabolism ; Humans ; Microbial Viability ; drug effects ; Microscopy, Confocal ; Polysaccharides, Bacterial ; biosynthesis ; Streptococcus mutans ; drug effects ; enzymology ; metabolism ; Sucrose ; metabolism ; pharmacology ; Sweetening Agents ; metabolism ; pharmacology ; Tetrazolium Salts ; Time Factors ; Up-Regulation ; Xylitol ; metabolism ; pharmacology

OBJECTIVETo invesitgate the expression patterns of amelogenin and enamelin in the developing tooth germs.

METHODSMandible sections of postnatal day 1, 3, 7 and 14 mouse were prepared, immunohistochemical analysis and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to detect the expression patterns of amelogenin and enamelin in mandibular first molars.

RESULTSAmelogenin was observed in the cytoplasm of secretory ameloblasts and the whole enamel matrix layer. It was also transiently expressed in the odontoblasts of postnatal day 1 molars. Enamelin proteins were observed in the enamel layer deposited by secretory ameloblasts, especially intense beneath the ameloblast process and dentino-enamel junction. The mRNA levels of both amelogenin and enamelin were highest on postnatal day 7 (the ratio to glyceraldehyde phosphate dehydrogenase of amelogenin and enamelin: 0.813 ± 0.085 and 0.799 ± 0.064, respectively, P < 0.05).

CONCLUSIONSAmelogenin and enamelin were enamel matrix proteins predominately expressed by secretory ameloblasts. The temporal-spatial expression patterns of amelogenin and enamelin indicate the important roles they played in amelogenesis and biomineralization.

Ameloblasts ; metabolism ; Amelogenesis ; Amelogenin ; genetics ; metabolism ; Animals ; Dental Enamel ; metabolism ; Dental Enamel Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred ICR ; Molar ; metabolism ; Odontoblasts ; metabolism ; RNA, Messenger ; metabolism ; Time Factors ; Tooth Germ ; growth & development ; metabolism

Human oral bacteria live in multispecies communities in the biofilm called dental plaque. This review focuses on the interactions of seven species and the ability of each species individually and together with other species to grow on saliva as the sole source of nutrient. Community formation in biofilms in flow cells is monitored using species-specific fluorophore-conjugated immunoglobulin G, and images are captured by confocal microscopy. Early colonizing veillonellae emerge from this review of interspecies interactions in saliva as a critical genus that guides the development of multispecies communities. Highly selective interspecies recognition is evident as initial colonizers pair with early and middle colonizers to form multispecies communities that grow on saliva.
Actinomyces ; growth & development ; Aggregatibacter actinomycetemcomitans ; growth & development ; Animals ; Biofilms ; growth & development ; Dental Enamel ; microbiology ; Dental Plaque ; metabolism ; microbiology ; Fluorescent Dyes ; metabolism ; Fusobacterium nucleatum ; growth & development ; Humans ; Microbial Consortia ; physiology ; Microbial Interactions ; physiology ; Microscopy, Confocal ; Polysaccharides, Bacterial ; chemistry ; Saliva ; metabolism ; microbiology ; Streptococcus oralis ; growth & development ; Veillonella ; growth & development

OBJECTIVEThere is a paucity of published works that systematically evaluate gene anomalies or clinical features of patients with renal cysts and diabetes syndrome (RCAD)/maturity onset diabetes of the young type 5 (MODY5). The purpose of this review was to systematically assess the detection rate, genetic and phenotypic implications of heterozygous autosomal dominant TCF2 anomalies.

DATA SOURCESMEDLINE database was searched to select articles recorded in English from 1997 to 2008. The focus was monoallelic germline TCF2 gene mutations/deletions. Biallelic inactivation, polymorphisms, DNA modification (hypomethylation and hypermethylation), loci associated with cancer risk, and somatic TCF2 anomalies were all excluded.

STUDY SELECTIONAfter searching the literature, 50 articles were selected.

RESULTSThe detection rate of TCF2 anomalies was 9.7% and varied considerably among MODY (1.4%), renal structure anomalies (RSA) (21.4%) and RSA with MODY (41.2%) subgroups. Mutations were strikingly located within the DNA binding domain and varied among exons of the DNA binding domain: exons 2 and 4 were the hottest spots, while mutations were sporadically distributed in exon 3. The consistent phenotypes were RSA (89.6%) and diabetes mellitus (DM) (45.0%). However, the concurrence of RSA and DM was relatively low (27.5%), which hinders the optimal performance of genetic testing and obtainment of timely diagnosis. Other organ involvements were complementary and necessary for the early identification of patients with TCF2 anomalies. Analysis of phenotypes of TCF2 point mutations showed significant differences in the detection rates of RSA, impaired renal function (IRF) and DM according to mutation type but not mutation location.

CONCLUSIONThese valuable features of TCF2 anomalies that previously did not receive sufficient attention should not be neglected.

Central Nervous System Diseases ; metabolism ; Dental Enamel ; abnormalities ; metabolism ; Diabetes Mellitus ; metabolism ; Diabetes Mellitus, Type 2 ; metabolism ; Hepatocyte Nuclear Factor 1-beta ; metabolism ; Humans ; Kidney Diseases, Cystic ; metabolism

This study sought to determine the impact of dental fluorosis severity on demineralization and remineralization of human fluorosed teeth in vitro. Surface enamel microhardness was measured on the enamel blocks before and after demineralization and after remineralization. The results showed that after demineralization, the sequence of % Surface microhardness demineralization (% SMHD) was TFI4 (18.92 +/- 1.31) < TFI3 (20.50 +/- 1.32) < TFI2 (25.08 +/- 1.69) < TFI1 (27.77 +/- 1.79) < TFI0 (30.70 +/- 1.35) (P < 0.05), and there was no statistically significant differences between TFI1 (27.77 +/- 1.79) and the normal group TFI0 (30.70 +/- 1.35). After remineralization, the sequence of % Surface microhardness remineralization (% SMHR) was TFI1 (55.17 +/- 1.23) > TFI0 (53.97 +/- 3.05) > TFI2 (49.17 +/- 1.81) > TFI3 (44.85 +/- 1.89) > TFI4 (36.51 +/- 2.95) (P < 0.05). Moderately fluorosed enamel showed a significatnt resistance to caries, but mildly fluorosed enamel could get better remineralization. These facts and figures deserve clinicians' attention.
Dental Enamel ; chemistry ; pathology ; Fluorosis, Dental ; metabolism ; Humans ; In Vitro Techniques ; Tooth Demineralization ; Tooth Remineralization

OBJECTIVETo investigate the adsorption behavior of enamel matrix proteins (EMPs) on the enamel surface and study their effect on biomineralization of enamel using quartz crystal microbalance (QCM) technique.

METHODS AND RESULTSThe EMPs were adsorbed on the enamel surface to form a protein film, which was soaked in simulated body fluid solutions. After 30 days of biomimetic mineralization, the hydroxyapatite nucleation, growth and aggregation occurred with hydroxyapatite crystal formation on the enamel surface.

CONCLUSIONThe EMPs play a key role in regulating enamel mineralization.

Adsorption ; Animals ; Dental Enamel Proteins ; metabolism ; pharmacology ; Humans ; Hydroxyapatites ; analysis ; chemistry ; Quartz ; Rats ; Rats, Sprague-Dawley ; Surface Properties ; Tooth Remineralization ; methods