ASH2L, Core Subunit of H3K4 Methylation Complex, Regulates Amelogenesis.

Histone methylation assumes a crucial role in the intricate process of enamel development. Our study has illuminated the substantial prevalence of H3K4me3 distribution, spanning from the cap stage to the late bell stage of dental germs. In order to delve into the role of H3K4me3 modification in amelogenesis and unravel the underlying mechanisms, we performed a conditional knockout of Ash2l, a core subunit essential for the establishment of H3K4me3 within the dental epithelium of mice. The absence of Ash2l resulted in reduced H3K4me3 modification, subsequently leading to abnormal morphology of dental germ at the late bell stage. Notably, knockout of Ash2l resulted in a loss of polarity in ameloblasts and odontoblasts. The proliferation and apoptosis of the inner enamel epithelium cells underwent dysregulation. Moreover, there was a notable reduction in the expression of matrix-related genes, Amelx and Dspp, accompanied with impaired enamel and dentin formation. Cut&Tag-seq (cleavage under targets and tagmentation sequencing) analysis substantiated a reduction of H3K4me3 modification on Shh, Trp63, Sp6, and others in the dental epithelium of Ash2l knockout mice. Validation through real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence consistently affirmed the observed downregulation of Shh and Sp6 in the dental epithelium following Ash2l knockout. Intriguingly, the expression of Trp63 isomers, DNp63 and TAp63, was perturbed in Ash2l defect dental epithelium. Furthermore, the downstream target of TAp63, P21, exhibited aberrant expression within the cervical loop of mandibular first molars and incisors. Collectively, our findings suggest that ASH2L orchestrates the regulation of crucial amelogenesis-associated genes, such as Shh, Trp63, and others, by modulating H3K4me3 modification. Loss of ASH2L and H3K4me3 can lead to aberrant differentiation, proliferation, and apoptosis of the dental epithelium by affecting the expression of Shh, Trp63, and others genes, thereby contributing to the defects of amelogenesis.

Adult dental epithelial stem cell-derived organoids deposit hydroxylapatite biomineral.

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.

Autoimmune amelogenesis imperfecta in patients with APS-1 and coeliac disease.

Ameloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation-amelogenesis1. Amelogenesis depends on multiple ameloblast-derived proteins that function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta2. Defects in enamel formation are also found in patients with autoimmune polyglandular syndrome type-1 (APS-1), caused by AIRE deficiency3,4, and in patients diagnosed with coeliac disease5-7. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta.

Evaluation of method parameters for sound undecalcified dental enamel proteomics using liquid chromatography-mass spectrometry.

OBJECTIVE: This study aims to validate a methodology for analyzing undecalcified, sound dental enamel proteomics using Liquid Chromatography-Mass Spectrometry (LC-MS). The study evaluates various parameters, including the impact of dental root coverage on protein contamination, the efficacy of protease inhibitors during enamel sample preparation, repeatability of LC-MS measurements on dental enamel, and statistical analysis. The study also assesses the effectiveness of combined trypsin and semi-trypsin searches in Mascot for obtaining additional protein identification data. DESIGN: Sound dental enamel was removed using a wet grinding technique, then digested with trypsin and labeled with TMT prior to LC-MS analysis. The resulting proteomes were matched against the Homo sapiens Swissprot Database, with searches in Mascot performed using both trypsin and semitrypsin. Statistical methods were employed to analyze the data. RESULTS: The study found that covering dental roots with composite during dental enamel microdissection is advisable, while using protease inhibition during microdissection may not be fully supported. The proteomic analyses demonstrated statistical repeatability and reliability, with consistent and reproducible proteomic data obtained from healthy dental enamel. Furthermore, employing both trypsin and semitrypsin searches in Mascot provided additional proteomic information. CONCLUSIONS: Overall, this study validates a methodology for analyzing undecalcified, sound dental enamel proteomics using LC-MS, and provides insights into various factors that can affect the quality and reliability of proteomic data. These findings have implications for future studies pursuant to understanding the proteomic mechanisms underlying dental enamel formation and other associated processes.

Tmem2 Deficiency Leads to Enamel Hypoplasia and Soft Enamel in Mouse.

Teeth consist of 3 mineralized tissues: enamel, dentin, and cementum. Tooth malformation, the most common craniofacial anomaly, arises from complex genetic and environmental factors affecting enamel structure, size, shape, and tooth eruption. Hyaluronic acid (HA), a primary extracellular matrix component, contributes to structural and physiological functions in periodontal tissue. Transmembrane protein 2 (TMEM2), a novel cell surface hyaluronidase, has been shown to play a critical role during embryogenesis. In this study, we demonstrate Tmem2 messenger RNA expression in inner enamel epithelium and presecretory, secretory, and mature ameloblasts. Tmem2 knock-in reporter mice reveal TMEM2 protein localization at the apical and basal ends of secretory ameloblasts. Micro-computed tomography analysis of epithelial-specific Tmem2 conditional knockout (Tmem2-CKO) mice shows a significant reduction in enamel layer thickness and severe enamel deficiency. Enamel matrix protein expression was remarkably downregulated in Tmem2-CKO mice. Scanning electron microscopy of enamel from Tmem2-CKO mice revealed an irregular enamel prism structure, while the microhardness and density of enamel were significantly reduced, indicating impaired ameloblast differentiation and enamel matrix mineralization. Histological evaluation indicated weak adhesion between cells and the basement membrane in Tmem2-CKO mice. The reduced and irregular expressions of vinculin and integrin ß1 suggest that Tmem2 deficiency attenuated focal adhesion formation. In addition, abnormal HA accumulation in the ameloblast layer and weak claudin 1 immunoreactivity in Tmem2-CKO mice indicate impaired tight junction gate function. Irregular actin filament assembly was also observed at the apical and basal ends of secretory ameloblasts. Last, we demonstrated that Tmem2-deficient mHAT9d mouse ameloblasts exhibit defective adhesion to HA-containing substrates in vitro. Collectively, our data highlight the importance of TMEM2 in adhesion to HA-rich extracellular matrix, cell-to-cell adhesion, ameloblast differentiation, and enamel matrix mineralization.

Peptide analysis of tooth enamel - A sex estimation tool for archaeological, anthropological, or forensic research.

Proteomics has become an attractive method to study human and animal material, biological profile, and origin as an alternative to DNA analysis. It is limited by DNA amplification in ancient samples and its contamination, high cost, and limited preservation of nuclear DNA. Currently, three approaches are available to estimate sex-osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. Proteomics provides a new, seemingly simple, and relatively non-expensive way of sex estimation without the risk of contamination. Proteins can be preserved in hard teeth tissue (enamel) for tens of thousands of years. It uses two sexually distinct forms of the protein amelogenin in tooth enamel detectable by liquid chromatography-mass spectrometry; the protein amelogenin Y isoform is present in enamel dental tissue only in males, while amelogenin isoform X can be found in both sexes. From the point of view of archaeological, anthropological, and forensic research and applications, the reduced destruction of the methods used is essential, as well as the minimum requirements for sample size.

Cuspal Shape Alterations by Bmp4 Directing Cell Proliferation and Apoptosis.

The enamel knot (EK), located at the center of cap stage tooth germs, is a transitory cluster of nondividing epithelial cells. The EK acts as a signaling center that provides positional information for tooth morphogenesis and regulates the growth of tooth cusps. To identify species-specific cuspal patterns, this study analyzed the cellular mechanisms in the EK that were related to bone morphogenetic protein (Bmp), which plays a crucial role in cell proliferation and apoptosis. To understand the cellular mechanisms in the EK, the differences between 2 species showing different cuspal patterning, mouse (pointy bunodont cusp) and gerbil (flat lophodont cusp), were analyzed with quantitative reverse transcriptase polymerase chain reaction and immunofluorescent staining. Based on these, we performed protein-soaked bead implantation on tooth germs of the 2 different EK regions and compared the cellular behavior in the EKs of the 2 species. Many genes related with cell cycle, cell apoptosis, and cell proliferation were involved in BMP signaling in the EK during tooth development. A comparison of the cell proliferation and apoptosis associated with Bmp revealed distinctive patterns of the cellular mechanisms. Our findings indicate that the cellular mechanisms, such as cell proliferation and apoptosis, in the EK are related to Bmp4 and play an important role in tooth morphogenesis.

Enamel Fluoride Uptake Determined Using the Microbiopsy Technique and Time-of-Flight Secondary Ion Mass Spectrometry: A Pilot Study.

This study evaluated the suitability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to assess enamel fluoride uptake (EFU) in comparison with the microbiopsy technique. Enamel specimens were exposed to equimolar solutions of fluoride prepared from sodium fluoride (NaF), stannous fluoride (SnF2), or amine fluoride (AmF). EFU was quantified by both techniques on the same specimens. EFU was found to be highest for samples treated with AmF, followed by SnF2 and NaF. Both methods yielded clearly interpretable, highly correlating (r = 0.95) data. ToF-SIMS can be considered a promising alternative to the microbiopsy technique for near-surface EFU assessment.

Tooth enamel nitrogen isotope composition records trophic position: a tool for reconstructing food webs.

Nitrogen isotopes are widely used to study the trophic position of animals in modern food webs; however, their application in the fossil record is severely limited by degradation of organic material during fossilization. In this study, we show that the nitrogen isotope composition of organic matter preserved in mammalian tooth enamel (δ15Nenamel) records diet and trophic position. The δ15Nenamel of modern African mammals shows a 3.7‰ increase between herbivores and carnivores as expected from trophic enrichment, and there is a strong positive correlation between δ15Nenamel and δ15Nbone-collagen values from the same individuals. Additionally, δ15Nenamel values of Late Pleistocene fossil teeth preserve diet and trophic level information, despite complete diagenetic loss of collagen in the same specimens. We demonstrate that δ15Nenamel represents a powerful geochemical proxy for diet that is applicable to fossils and can help delineate major dietary transitions in ancient vertebrate lineages.

Mechanistic Insights into Bioengineered Antibiofilm Enamel Pellicles.

Dental caries remains the most widespread chronic disease worldwide. Basically, caries originates within biofilms accumulated on dental enamel. Despite the nonrenewable nature of the enamel tissue, targeted preventive strategies are still very limited. We previously introduced customized multifunctional proteinaceous pellicles (coatings) for controlling bacterial attachment and subsequent biofilm succession. Stemmed from our whole proteome/peptidome analysis of the in vivo acquired enamel pellicle, we designed these pellicles using hybrid mixtures of the most abundant and complementary-acting antimicrobial and antifouling proteins/peptides for synergetic suppression of early biofilms. In conjugating these domains synthetically, their bioinhibitory efficacy was remarkably boosted. Herein, we sought to explore the key structure-function relationship of these potent de novo hybridized conjugates in comparison with their individual domains, solely or in physical mixtures. Specifically, we interrelated the following facets: physicochemical and 3-dimensional folding characteristics via molecular dynamics simulations, adopted secondary structure by circular dichroism, immobilization capacity on enamel through high-spatial resolution multiphoton microscopy, and biofilm suppression potency. Our data showed consistent associations among the increased preference for protein folding structures, α-helix content, and enamel-immobilization capacity; all were inversely correlated with the attached bioburden. The expressed phenotypes could be explained by the adopted strongly amphipathic helical conformation upon conjugation, mediated by the highly anionic and acidic N-terminal pentapeptide shared region/motif for enhanced immobilization on enamel. In conclusion, conjugating bioactive proteins/peptides is a novel translational approach to engineer robust antibiofilm pellicles for caries prevention. The adopted α-helical conformation is key to enhance the antibiofilm efficacy and immobilization capacity on enamel that are promoted by certain physicochemical properties of the constituent domains. These data are valuable for bioengineering versatile therapeutics to prevent/arrest dental caries, a condition that otherwise requires invasive treatments with substantial health care expenditures.

Conditional knockout of transient receptor potential melastatin 7 in the enamel epithelium: Effects on enamel formation.

Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.

Overexpression of RCAN1, a Gene on Human Chromosome 21, Alters Cell Redox and Mitochondrial Function in Enamel Cells.

The regulator of calcineurin (RCAN1) has been implicated in the pathogenesis of Down syndrome (DS). Individuals with DS show dental abnormalities for unknown reasons, and RCAN1 levels have been found to be elevated in several tissues of DS patients. A previous microarray analysis comparing cells of the two main formative stages of dental enamel, secretory and maturation, showed a significant increase in RCAN1 expression in the latter. Because the function of RCAN1 during enamel formation is unknown, there is no mechanistic evidence linking RCAN1 with the dental anomalies in individuals with DS. We investigated the role of RCAN1 in enamel by overexpressing RCAN1 in the ameloblast cell line LS8 (LS8+RCAN1). We first confirmed that RCAN1 is highly expressed in maturation stage ameloblasts by qRT-PCR and used immunofluorescence to show its localization in enamel-forming ameloblasts. We then analyzed cell redox and mitochondrial bioenergetics in LS8+RCAN1 cells because RCAN1 is known to impact these processes. We show that LS8+RCAN1 cells have increased reactive oxygen species (ROS) and decreased mitochondrial bioenergetics without changes in the expression of the complexes of the electron transport chain, or in NADH levels. However, LS8+RCAN1 cells showed elevated mitochondrial Ca2+ uptake and decreased expression of several enamel genes essential for enamel formation. These results provide insight into the role of RCAN1 in enamel and suggest that increased RCAN1 levels in the ameloblasts of individuals with DS may impact enamel formation by altering both the redox environment and mitochondrial function, as well as decreasing the expression of enamel-specific genes.

Odontogenesis-Associated Phosphoprotein (ODAPH) Overexpression in Ameloblasts Disrupts Enamel Formation via Inducing Abnormal Mineralization of Enamel in Secretory Stage.

Odontogenesis-associated phosphoprotein (ODAPH) is a recently discovered enamel matrix protein. Our previous study demonstrated that knockouting out Odaph in mice resulted in enamel hypomineralization. To further investigate the effect of Odaph on enamel mineralization, we constructed an Odaph overexpression mouse model, controlled by an amelogenin promoter. Our histological analysis of OdaphTg mice revealed that the enamel layer was thinner than in WT mice. An uneven, thinner enamel layer was confirmed using micro-computed tomography (uCT). It was subsequently found that the Tomes' processes lost their normal morphology, resulting in the loss of the enamel prism structure. These results indicate that Odaph overexpression in ameloblasts led to enamel dysplasia. In conjunction with this, Odaph overexpression hindered Amelx secretion, and may result in endoplasmic reticulum stress. Interestingly, uCT revealed that enamel had higher mineral density at the secretory stage; due to this, we did the histological staining for the mineralization-related proteins Alkaline phosphatase (ALPL) and Runt-related transcription factor 2 (RUNX2). It was observed that these proteins were up-regulated in OdaphTg mice versus WT mice, indicating that Odaph overexpression led to abnormal enamel mineralization. To confirm this, we transfected ameloblast-like cell line (ALC) with Odaph overexpression lentivirus in vitro and identified that both Alpl and Runx2 were strikingly upregulated in OE-mus-Odaph versus OE-NC cells. We concluded that the ectopic overexpression of Odaph in ameloblasts led to abnormal enamel mineralization. In summary, Odaph profoundly influences amelogenesis by participating in enamel mineralization.

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.

Self-assembly peptide P11-4 induces mineralization and cell-migration of odontoblast-like cells.

OBJECTIVES: Self-assembling peptide P11-4 is amphiphilic and pH-triggered, effective on repairing early enamel carious lesions and dentin remineralization. However, P11-4 effects on dentin biomineralization and repair ability remain unexplored. Thus, cytocompatibility and effectiveness of P11-4 on inducing mineralization and migration of odontoblast-like cells (MDPC-23) were investigated. METHODS: MDPC-23 were seeded in contact with P11-4 (0.5 and 1 µg/ml), Dentin Matrix Protein 1 (DMP1 0.5 and 1 µg/ml) or Calcium hydroxide (Ca(OH)2 100 µg/ml) solutions. Cell viability was verified using MTT (n = 6/group). Mineral deposition was tested using Alizarin Red (n = 4/group). Cell migration was assessed by light microscopy (n = 2/group). MTT and Alizarin Red data were compared using Kruskal-Wallis and Mann-Whitney (α=0.01). RESULTS: P11-4 (0.5 and 1 µg/ml) and DMP1 (0.5 and 1 µg/ml) resulted the highest cell viability; Ca(OH)2 presented the lowest. 1 µg/ml DMP1 and 1 µg/ml P11-4 promoted the highest mineral deposition. Ca(OH)2 presented lower values of mineral deposits than DMP1 1 µg/ml (p < 0.01), but similar to P11-4 1 µg/ml. P11-4 and DMP1 at 0.5 µg/ml induced lesser mineral precipitation than P11-4 and DMP1 at 1 µg/ml (p < 0.01), with no difference to Ca(OH)2. All materials stimulated cell migration, however, lower concentrations of DMP1 and P11-4 demonstrated a higher migration potential. CONCLUSION: P11-4 did not affect cell viability, induces mineral deposition and MDPC-23 migration like DMP1. CLINICAL SIGNIFICANCE: Self-assembling peptide P11-4 does not affect the cell viability and induces mineral deposition comparable to native protein involved in biomineralization. Combined with its ability to bind type I collagen, P11-4 is a promising bioinspired molecule that provides native-tissue conditions and foster further studies on its ability to form dentin bridges in pulp-capping strategies.

WDR72 regulates vesicle trafficking in ameloblasts.

As the hardest tissue in the human body, tooth enamel formation is a highly regulated process involving several stages of differentiation and key regulatory genes. One such gene, tryptophan-aspartate repeat domain 72 (WDR72), has been found to cause a tooth enamel defect when deleted or mutated, resulting in a condition called amelogenesis imperfecta. Unlike the canonical genes regulating tooth development, WDR72 remains intracellularly and is not secreted to the enamel matrix space to regulate mineralization, and is found in other major organs of the body, namely the kidney, brain, liver, and heart. To date, a link between intracellular vesicle transport and enamel mineralization has been suggested, however identification of the mechanistic regulators has yet to be elucidated, in part due to the limitations associated with studying highly differentiated ameloblast cells. Here we show compelling evidence that WDR72 regulates endocytosis of proteins, both in vivo and in a novel in vitro ameloblast cell line. We elucidate WDR72's function to be independent of intracellular vesicle acidification while still leading to defective enamel matrix pH extracellularly. We identify a vesicle function associated with microtubule assembly and propose that WDR72 directs microtubule assembly necessary for membrane mobilization and subsequent vesicle transport. Understanding WDR72 function provides a mechanistic basis for determining physiologic and pathologic tissue mineralization.

Phosphorylated and Phosphonated Low-Complexity Protein Segments for Biomimetic Mineralization and Repair of Tooth Enamel.

Biomimetic mineralization based on self-assembly has made great progress, providing bottom-up strategies for the construction of new organic-inorganic hybrid materials applied in the treatment of hard tissue defects. Herein, inspired by the cooperative effects of key components in biomineralization microenvironments, a new type of biocompatible peptide scaffold based on flexibly self-assembling low-complexity protein segments (LCPSs) containing phosphate or phosphonate groups is developed. These LCPSs can retard the transformation of amorphous calcium phosphate into hydroxyapatite (HAP), leading to merged mineralization structures. Moreover, the application of phosphonated LCPS over phosphorylated LCPS can prevent hydrolysis by phosphatases that are enriched in extracellular mineralization microenvironments. After being coated on the etched tooth enamel, these LCPSs facilitate the growth of HAP to generate new enamel layers comparable to the natural layers and mitigate the adhesion of Streptococcus mutans. In addition, they can effectively stimulate the differentiation pathways of osteoblasts. These results shed light on the potential biomedical applications of two LCPSs in hard tissue repair.

Validation of a cariogenic biofilm model by evaluating the effect of fluoride on enamel demineralization.

In vitro biofilm models have been extensively used, but only few of the models available to date had been validated in terms of the dose-response effect of anti-caries and/or antimicrobial substances. Additionally, none of the validated models allow the use of microliter volumes of the treatment solutions, needed mainly to test (screen) novel but expensive substances under development. This study aimed at modifying an in vitro cariogenic Streptococcus mutans biofilm model and validating it by assessing the dose-response effect of fluoride on enamel demineralization. S. mutans cariogenic biofilms were developed on saliva-coated enamel slabs previously bonded to acrylic holders fixed to a lid of a culture plate. Biofilms were incubated 8 h/day in culture medium supplemented with 1% sucrose and then overnight in culture medium with glucose 0.1 mM. Biofilms were also treated 2×/day with 2.0 mL of solutions containing 0, 125, 275 and 1250 µg F/mL (n = 10/group). The replaced culture medium was used to: determine the biofilm acidogenicity; estimate the demineralization of enamel; and monitor the fluoride concentration. At 144 h, biofilms were collected for fluoride concentration analyses, and the fluoride uptake by enamel was determined in each slab. The model showed a dose-response effect of fluoride (R2 = 0.96, p < 0.001) between enamel demineralization and the fluoride concentration of the treatments. Water-soluble and bound biofilm fluoride concentrations (p < 0.007), as well as the firmly-bound fluoride concentration found in enamel (p < 0.0001), increased in a dose-dependent manner. Our model constitutes a validated approach that would allow the assessment of the anticaries potential of novel biotechnological strategies, as in the case of expensive salivary peptides, because it would allow to test the treatment solutions using smaller volumes.

Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel.

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed "shedding", while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel "dahlite" crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.

Mouse Dspp frameshift model of human dentinogenesis imperfecta.

Non-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein (DSPP): 5' mutations affecting an N-terminal targeting sequence and 3' mutations that shift translation into the - 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp-1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a DsppP19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. DsppP19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. DsppP19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp-1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp+/+ and DsppP19L dentin. The Dspp-1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5' and 3' Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.