The circadian clock in enamel development

Circadian rhythms are self-sustaining oscillations within biological systems that play key roles in a diverse multitude of physiological processes.The circadian clock mechanisms in brain and peripheral tissues can oscillate independently or be synchronized/disrupted by external stimuli.Dental enamel is a type of mineralized tissue that forms the exterior surface of the tooth crown.Incremental Retzius lines are readily observable microstructures of mature tooth enamel that indicate the regulation of amelogenesis by circadian rhythms.Teeth enamel is formed by enamel-forming cells known as ameloblasts,which are regulated and orchestrated by the circadian clock during amelogenesis.This review will first examine the key roles of the circadian clock in regulating ameloblasts and amelogenesis.Several physiological processes are involved,including gene expression,cell morphology,metabolic changes,matrix deposition,ion transportation,and mineralization.Next,the potential detrimental effects of circadian rhythm disruption on enamel formation are discussed.Circadian rhythm disruption can directly lead to Enamel Hypoplasia,which might also be a potential causative mechanism of amelogenesis imperfecta.Finally,future research trajectory in this field is extrapolated.It is hoped that this review will inspire more intensive research efforts and provide relevant cues in formulating novel therapeutic strategies for preventing tooth enamel developmental abnormalities.

Research on the Application of Contemporary Wireless Enamel Technique in the Teaching of Traditional Jewelry Craft Course for College Student

Taking the wireless enamel(wireless enamel refers to make enamel works without filigree)production technique in jewelry practice course as the research object,combined with years of enamel teaching experience in the Gemmological Institute of China University of Geosciences(Wuhan),this study expounds the theoretical class,design preparation,creative idea and technique practice application from the course teaching objectives and the implementation,which also emphasizes the practical improvability of students’design practice ability and innovative thinking ability.During the period,it focuses on the core processes,such as wireless enamel glaze method,object correction,firing time,composite application and system integration,to help students solve the key and difficult problems in the wireless enamel firing process.

The importance of a potential phosphorylation site in enamelin on enamel formation

Enamelin （ENAM） has three putative phosphoserines （pSers） phosphorylated by a Golgi-associated secretory pathway kinase （FAM20C） based on their distinctive Ser-x-Glu （S-x-E） motifs. Fam2OC-knockout mice show severe enamel defects similar to those in the Enam-knockout mice, implying an important role of the pSers in ENAM. To determine the role of pSer5s in ENAM, we characterized ENAMRgsc514 mice, in which Sers5 cannot be phosphorylated by FAM20C due to an E57〉Gs7 mutation in the S-x-E motif, The enamel microstructure of 4-week-old mice was examined by scanning electron microscopy. The teeth of 6-day-old mice were characterized by histology and immunohistochemistry. The protein lysates of the first lower molars of 4-day-old mice were analyzed by Western immunoblotting using antibodies against ENAM, ameloblastin and amelogenin. ENAMRgsc514 heterozygotes showed a disorganized enamel microstructure, while the homozygotes had no enamel on the dentin surface. The N-terminal fragments of ENAM in the heterozygotes were detained in the ameloblasts and localized in the mineralization front of enamel matrix, while those in the WT mice were secreted out of ameloblasts and distributed evenly in the outer 1/2 of enamel matrix. Surprisingly, the 15 kDa C-terminal fragments of ameloblastin were not detected in the molar lysates of the homozygotes. These results suggest that the phosphorylation of SerSS may be an essential posttranslational modification of ENAM and is required for the interaction with other enamel matrix molecules such as ameloblastin in mediating the structural organization of enamel matrix and protein-mineral interactions during enamel formation.

Deformation prediction of porcelain-enameled steels with strain history by press forming and high-temperature behavior of coating layer

Porcelain enamel coatings were widely applied for the protection of steel products because they offered high corrosion protection,resistance to heat and abrasion,high hardness,hygiene and ease of cleaning.The typical process to produce enameled steels is roughly divided into two stages:the first stage consists of a forming process to give the desired shape to a steel substrate,and the second stage consists of a firing process to bond enamel frits on the substrate.This firing process requires a high temperature above 800 °C,which may lead to austenitic transformation and severe thermal deformation of the steel substrate.The aim of this study is to develop a finite element analysis (FE analysis) technique to predict the mechanical and thermal deformations of the enameled steels during forming and any further enameling process,including firing.The FE analysis involves analyzing the strain history of the steel substrate,which comprises the stress and thickness distributions of the substrate and its deformed shape,and the high-temperature behavior of the enamel coating layer.The validity of the FE analysis is verified through the U-bending test and firing test with various numbers and positions of enamel coating layers on the substrate.The results reveal that the FE analysis results agree well with the experimental results with 8% error.

Causes of the formation of pit defects on the surface of the enamel layer

During the production of the inner walls of ovens,pit defects were formed on the surface of the enamel layer that was enameled on the cold-rolled steel via electrostatic powder spraying and sintering.The paper elaborates on the microstructure and element distribution of the enamel-steel interface and the enamel layer.Optical microscopy,scanning electron microscopy,and energy-dispersive spectroscopy were adopted to investigate the microstructure of a longitudinal section of the defect,and the pit-forming causes were analyzed.The results show that rusty spots lead to pit defects.During high-temperature firing,there is an inadequate fusion and reaction between iron oxides of the rusty spots and the enamel glaze.The rusty spots are closely related to pretreatment process;thus,to avoid their occurrence,electrostatic powder spraying and sintering should be performed timely after forming,degreasing,and thorough drying of the metal sheets.

Enamel microabrasion: An overview of clinical and scientific considerations

Superficial stains and irregularities of the enamelare generally what prompt patients to seek dental intervention to improve their smile. These stains or defects may be due to hypoplasia, amelogenesis imperfecta, mineralized white spots, or fluorosis, for which enamel microabrasion is primarily indicated. Enamel microabrasion involves the use of acidic and abrasive agents, such as with 37% phosphoric acid and pumice or 6% hydrochloric acid and silica, applied to the altered enamel surface with mechanical pressure from a rubber cup coupled to a rotatory mandrel of a lowrotation micromotor. If necessary, this treatment can be safely combined with bleaching for better esthetic results. Recent studies show that microabrasion is a conservative treatment when the enamel wear is minimal and clinically imperceptible. The most important factor contributing to the success of enamel microabrasion is the depth of the defect, as deeper, opaque stains, such as those resulting from hypoplasia, cannot be resolved with microabrasion, and require a restorative approach. Surface enamel alterations that result from microabrasion, such as roughness and microhardness, are easily restored by saliva. Clinical studies support the efficacy and longevity of this safe and minimally invasive treatment. The present article presents the clinical and scientific aspects concerning the microabrasion technique, and discusses the indications for and effects of the treatment, including recent works describing microscopic and clinical evaluations.

Microstructure and properties of Ti-bearing hot-rolled high-strength steel plates for two-sided enameling

The composition and production technology of the type of hot-rolled steel plate used in two-sided enameling were briefly described. The microstructure and mechanical properties before and after enameling were contrastively investigated,and the precipitates in the samples were analyzed using transmission electron microscope and energy dispersive spectrometer. The results show the ferrite grain size of the steel plate after high-temperature enamel firing to be fine,with a large number of TiC and Ti;C;S;precipitates dispersed throughout the ferrite matrix. After two rounds of enamel firing at a temperature range of 800-890 ℃,its yield strength can still reach342 MPa. The results of a hydrogen permeation test show that the hydrogen storage properties of the steel plate are much better than those of ordinary structural carbon steel. A better bubble structure in the enamel layer can be obtained by this steel plate,with no fish-scale defects on the enameled steel-plate surface.

Effects of 45S5 bioglass on surface properties of dental enamel subjected to 35% hydrogen peroxide

Tooth bleaching agents may weaken the tooth structure. Therefore, it is important to minimize any risks of tooth hard tissue damage caused by bleaching agents. The aim of this study was to evaluate the effects of applying 45S5 bioglass （BG） before, after, and during 35% hydrogen peroxide （HP） bleaching on whitening efficacy, physicochemical properties and microstructures of bovine enamel. Seventy-two bovine enamel blocks were prepared and randomly divided into six groups： distilled deionized water （DDW）, BG, HP, BG before HP, BG after HP and BG during HP. Colorimetric and microhardness tests were performed before and after the treatment procedure. Representative specimens from each group were selected for morphology investigation after the final tests. A significant color change was observed in group HP, BG before HP, BG after HP and BG during HP. The microhardness loss was in the following order： group HP〉 BG before HP, BG after HP〉 BG during HP〉DDW, BG. The most obvious morphological alteration of was observed on enamel surfaces in group HP, and a slight morphological alteration was also detected in group BG before HP and BG after HP. Our findings suggest that the combination use of BG and HP could not impede the tooth whitening efficacy. Using BG during HP brought better protective effect than pre/post-bleaching use of BG, as it could more effectively reduce the mineral loss as well as retain the surface integrity of enamel. BG may serve as a promising biomimetic adjunct for bleaching therapy to prevent/restore the enamel damage induced by bleaching agents.

The effect of cold-light-activated bleaching treatment on enamel surfaces in vitro

This in vitrostudy aims to evaluate the crystal and surface microstructure of dental enamel after cold-light bleaching treatment. Twelve sound human premolars were cross-split into four specimens, namely, mesio-buccal （Group LP）, disto-buccal （Group P）, mesio-lingual （Group NP） and disto-lingual （Group L） specimens. These four groups were treated using the standard cold-light bleaching procedure, a bleaching agent, a peroxide-free bleaching agent and cold-light, respectively. Before and after treatment, all specimens were analyzed by high-resolution, micro-area X-ray diffraction and scanning electron microscopy. Using a spectrometer, tooth color of all specimens was measured before and after treatment. The phase of the enamel crystals was identified as hydroxyapatite and carbonated hydroxyapatite. After treatment, specimens in Groups LP and P showed significantly weaker X-ray diffraction peaks, significant reduction in crystal size and crystallinity, significant increase in L~ but decrease in a＊ and b＊, and obvious alterations in the surface morphology. However, specimens in Groups NP and L did not show any significant changes. The cold-light bleaching treatment leads to demineralization in the enamel surface. The acidic peroxide-containing bleaching agent was the major cause of demineralization, whereas cold-light did not exhibit significant increase or decrease effect on this demineralization.

Effect of Galla chinensis on the In Vitro Remineralization of Advanced Enamel Lesions

Aim The effect of Galla chinensis on de-/re-mineralization of advanced enamel lesions was investigated by using micro-CT in a prolonged in vitro experiment. Methodology Baseline mineral contents of sound enamels were first analyzed. Then lesions were produced in an acidic buffer solution （2.2 mmol.L-1 Ca（NO3）2, 2.2 mmol-L1 KH2PO4, and pH=4.5） for 21 days, with thrice daily three-minute treatments, divided into four groups： Group A, 4 000 ppm crude aqueous extract of Galla chinensis （GCE）; Group B, 4 000 ppm gallic acid; Group C, 1 000 ppm F aq. （as NaF, positive control）; Group D, deionized water （negative control）. Next, the blocks were immersed in a remineralization solution （1.5 mmol.Lz CaC12, 0.9 mmol.L1 KH2PO4, 0.1 ppm F, and pH=7,0） for 200 days. Mineral loss （ML） in each region of interest （ROI） and integrated mineral loss （IML） of the lesions were calculated （comparing with baseline mineral content of sound enamel） at different time points. Results After 21 days demineralization, fluoride treatment showed a statistically significant demineralization-inhibiting effect among the four groups, and after 200 days of remineralization, mineral content recovery was ordered （lowest to highest） as A=C〈B〈D. Conclusion GCE could slow down the remineralization of enamel in the surface layer and thereby facilitate ion transport into the lesion body. The mechanism of Galla chinensis in enhancing the remineralization of dental caries is different from fluoride.

Human enamel thickness and ENAM polymorphism

The tooth enamel development gene, enamelin （ENAM）, showed evidence of positive selection during a genome-wide scan of human and primate DNA for signs of adaptive evolution. The current study examined the hypothesis that a single-nucleotide polymorphism （SNP） C14625T （rs7671281） in the ENAM gene identified in the genome-wide scan is associated with a change in enamel phenotype. African Americans were selected as the target population, as they have been reported to have a target SNP frequency of approximately 50%, whereas non-Africans are predicted to have a 96% SNP frequency. Digital radiographs and DNA samples from 244 teeth in 133 subjects were analysed, and enamel thickness was assessed in relation to SNP status, controlling for age, sex, tooth number and crown length. Crown length was found to increase with molar number, and females were found to have thicker enamel. Teeth with larger crowns also had thicker enamel, and older subjects had thinner enamel. Linear regression and generalized estimating equations were used to investigate the relationship between enamel thickness of the mandibular molars and ENAM SNP status; enamel in subjects with the derived allele was significantly thinner （P= 0.040） when the results were controlled for sex, age, tooth number and crown length. The derived allele demonstrated a recessive effect on the phenotype. The data indicate that thinner dental enamel is associated with the derived ENAM genotype. This is the first direct evidence of a dental gene implicated in human adaptive evolution as having a phenotypic effect on an oral structure.

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.

Dentin matrix protein 1 and phosphate homeostasis are critical for postnatal pulp, dentin and enamel formation

Deletion or mutation of dentin matrix protein 1 （DMP1） leads to hypophosphatemic rickets and defects within the dentin. However, it is largely unknown if this pathological change is a direct role of DMP1 or an indirect role of phosphate （Pi） or both. It has also been previously shown that Klotho-deficient mice, which displayed a high Pi level due to a failure of Pi excretion, causes mild defects in the dentinal structure. This study was to address the distinct roles of DMP1 and Pi homeostasis in cell differentiation, apoptosis and mineralization of dentin and enamel. Our working hypothesis was that a stable Pi homeostasis is critical for postnatal tooth formation, and that DMP1 has an antiapoptotic role in both amelogenesis and dentinogenesis. To test this hypothesis, Dmpl-null （Dmpl-/-）, Klotho-deficient （kl/kl）, Dmpl/Klotho-double-deficient （Dmpl-/-/kl/kl） and wild-type （WT） mice were killed at the age of 6 weeks. Combinations of X-ray, microcomputed tomography （I^CT）, scanning electron microscopy （SEM）, histology, apoptosis and immunohistochemical methods were used for characterization of dentin, enamel and pulp structures in these mutant mice. Our results showed that Dmpl-/- （a low Pi level） or kl/kl（a high Pi level） mice displayed mild dentin defects such as thin dentin and a reduction of dentin tubules. Neither deficient mouse line exhibited any apparent changes in enamel or pulp structure. However, the double-deficient mice （a high Pi level） displayed severe defects in dentin and enamel structures, including loss of dentinal tubules and enamel prisms, as well as unexpected ectopic ossification within the pulp root canal. TUNEL assay showed a sharp increase in apoptotic cells in ameloblasts and odontoblasts. Based on the above findings, we conclude that DMP1 has a protective role for odontoblasts and ameloblasts in a pro-apoptotic environment （a high Pi level）.

Effectiveness of some herbals on initial enamel caries lesion

Objective: To evaluate the effectiveness of herbal medicaments such as ginger, rosemary and honey on remineralization of initial enamel lesion.Methods: Demineralized human enamel specimens were measured for baseline surface microhardness and fluorescence methods. Ten specimens in each of four groups were used in this in vitro recycling study with the following treatments which applied three times a day: 1) sodium fluoride toothpaste(Ipana, Procter & Gamble, Turkey), 2) gingerhoney(Arifoglu Herbals, Anzer Honey, Turkey), 3) ginger-honey-chocolate(Bind Chocolate, Turkey), 4) rosemary oil(Arifoglu Herbals, Turkey). Treatment regimens of demineralization and remineralization cycle were applied for 21 days. The post-treatment data were obtained by measurements of surface microhardness and fluorescence methods.Data were statistically analyzed by ANOVA test with Tukey's honest significant difference test.Results: Enhanced remineralization was observed with several of the treatment systems including ginger + honey and rosemary. Significant differences between treatments were observed by microhardness and Fluore Cam fluorescence assesment, compared to the positive control group(Na F dentifrice). Significantly, greater remineralization was observed with the honey + ginger treatment regimen. No significant differences between groups were observed using the fluorescence assessment method, quantitative lightinduced fluorescence.Conclusions: Herbals(ginger, honey and rosemary) have enhanced remineralization of initial enamel lesion.

Region-dependent micro damage of enamel under indentation

The objective of this investigation is to explore the region-dependent damage behavior of enamel, as well as to develop a good understanding of the deformation mech- anisms of enamel with numerical modeling. Nanoinden- tation experiments have been performed to investigate the load-penetration depth responses for outer and inner enamel. Results show that the unloading curve does not follow the loading curve, and degradation of stiffness in the unloading curve is observed. Based on the experimental data, a physi- cal quantity, the chain density in protein, has been introduced to the Drucker-Prager plastic model. Numerical simulations show that the simulated load-penetration depth curves agree with the experiments, and the stiffness degradation behav- iors of outer and inner enamel are captured by the numerical model. The region-dependent damage behavior of enamel could be revealed by the numerical model. The micro dam- age affected area at inner enamel is larger than that at outer enamel, indicating that the inner enamel experiences more micro damage than the outer one. Compared with its outer counterpart, the inner enamel which is rich in organic protein could break more internal protein chains to dissipate energy and to enhance its resistance to fracture accordingly.

Effect of Coiling Temperature on Ti(C,N) Precipitation and Properties of Batch Annealed DC06EK Enamel Steel

The effects of hot-strip coiling temperature on Ti（C,N） precipitation, texture and hydrogen permeation behavior in DC06 EK enamel steel were investigated by TEM, EBSD test and electrochemical hydrogen permeation experiment. It was found that the Ti（C,N） particles in hot-strip coarsened with increasing coiling temperature, whereas after cold-rolling and annealing, the size difference of Ti（C,N） particles was lessened. The hot-strip coiling temperature has a significant impact on the recrystallized texture in the subsequent cold-rolled and annealed sheet. Hot-strip using high temperature（700 ℃） coiling leads to strong {111} recrystallized texture in annealed sheet, with peak intensity 9.2. On the contrary, in annealed sheets using hot-strip coiling at 650 ℃, their {111} recrystallized textures were weaker, which was also reflected in their rmvalues. Even though the hydrogen diffusion coefficient is slightly lower（7.76×10^（-5） mm^2/s） in annealed sheet using low temperature coiling（600 ℃）, appropriately higher coiling temperature is more suitable for DC06 EK enamel steel combining both good drawability and fish-scale resistance.

A simple,sensitive and non-destructive technique for characterizing bovine dental enamel erosion:attenuated total reflection Fourier transform infrared spectroscopy

Although many techniques are available to assess enamel erosion in vitro, a simple, non-destructive method with sufficient sensitivity for quantifying dental erosion is required. This study characterized the bovine dental enamel erosion induced by various acidic beverages in vitro using attenuated total reflection Fourier transform infrared （ATR-FTIR） spectroscopy. Deionized water （control） and 10 acidic beverages were selected to study erosion, and the pH and neutralizable acidity were measured. Bovine anterior teeth （110） were polished with up to 1 200-grit silicon carbide paper to produce flat enamel surfaces, which were then immersed in 20 mL of the beverages for 30 min at 37 ℃. The degree of erosion was evaluated using ATR-FTIR spectroscopy and Vickers＇ microhardness measurements. The spectra obtained were interpreted in two ways that focused on the ~1, ~3 phosphate contour： the ratio of the height amplitude of ~3 P04 to that of/11 P04 （Method 1） and the shift of the v3 P04 peak to a higher wavenumber （Method 2）. The percentage changes in microhardness after the erosion treatments were primarily affected by the pH of the immersion media. Regression analyses revealed highly significant correlations between the surface hardness change and the degree of erosion, as detected by ATR-FTIR spectroscopy （P〈0.001）. Method 1 was the most sensitive to these changes, followed by surface hardness change measurements and Method 2. This study suggests that ATR- FTIR spectroscopy is potentially advantageous over the microhardness test as a simple, non-destructive, sensitive technique for the quantification of enamel erosion.

Investigation of Adsorption of Xanthan Gum on Enamel by Tapping Mode Atomic Force Microscopy

By using tapping mode atomic force microscopy(TMAFM), a polymer layer was found on the enamel surface after the exposure to xanthan gum solutions. The layer thickness is closely related to the exposure time and the concentration of xanthan gum solution. The thickness data were evaluated by a Kruskal-Wallis test and Box-Whisker Plot at a 95% confidence level(p<0.05), and a statistically significant difference among the thickness data groups was demonstrated. After the exposure to 1000, 400, 100 mg/L xanthan gum solutions, the mean of layer thickness at the adsorption equilibrium is in the ranges of 103.5_122.06, 82.4_88.94 and 45.27_55.55 nm, respectively. This phenomenon suggests that the viscosity modifying agents in the beverage might be adsorbed on the enamel surface during consumption, which may form a barrier that can protect the enamel from being attacked by acid and therefore reduce dental erosion.

A Comparable Study of Combinational Regenerative Therapies Comprising Enamel Matrix Derivative plus Deproteinized Bovine Bone Mineral with or without Collagen Membrane in Periodontitis Patients with Intrabony Defects

Aim: The aim of the present study was to examine the effectiveness of collagen membrane (CM) in regenerative therapy with deproteinized bovine bone mineral (DBBM) and enamel matrix derivative (EMD) for periodontal intrabony defects. Methods: Eighteen periodontal intrabony defects of nine chronic periodontitis patients were evaluated. Two defects per patient with probing pocket depth (PPD) ≥ 6 mm were assigned to two different types of treatments: EMD + DBBM + CM or EMD + DBBM. Clinical parameters including Gingival Index (GI), PPD, clinical attachment level (CAL), gingival recession (GR), bleeding on probing (BOP), tooth mobility (MOB), and the filled bone volume/rate (FBV/FBR), which was measured by cone beam computed tomography, were compared at baseline and 12 months post-treatment. Differences between groups were determined by the chisquare test, McNemar’s test, and Wilcoxon signed-rank test. Results: Clinically, PPD, CAL, and FBR significantly improved in both groups (p Conclusion: Periodontal regenerative therapies comprising EMD and DBBM with and without CM resulted in positive clinical outcomes. The use of CM may result in better outcomes in MOB decrease;however, long-term prognosis must be further studied.

Bonding Efficacy of a Self-adhesive Resin Cement to Enamel and Dentin

With the objective to evaluate the bonding efficacy of a new self-adhesive resin cement（RelyX Unicem, 3M ESPE） to enamel and dentin using a shear bonding strengths test with or without acid etching pretreatment, flat buccal dentin surface and mesial/distal enamel surface were made using a high-speed diamond bur. Copper rings were luted using Rely X Unicem（RU; 3M ESPE）, Panavia F（PF; Kuraray） or Vitique（VI; DMG）. For RU, the shear bonding strengths using GL（Gluma Etch, Heraeus） acid etching pretreatment were also tested. The teeth were placed into copper rings（inner diameter： 16 mm, height： 4mm） and embedded in methylmethacrylate resin. The specimens were stored for 24 h in distilled water at 37 ℃ prior to shear bonding strengths testing. In addition, bond failures were examined by optical microscope and categorized as 4 models such as different adhesive, cohesive, or mixed. Shear bonding strengths were calculated by dividing the maximum debonding force over the cross sectional area of each specimen. The Kruskal-Wallis test was used to determine pairwise statistical differences（P 〈 0.05） in SBS between the experimental groups. For dentin bonding strength, statistical analysis showed that there was no significant difference among RU（12.84 MPa）, PF（14.93 MPa） and VI（11.03 MP）; and the bonding strengths of them were higher than RU with acid etching pretreatment（9.12 MP）. When bonded to enamel, PF（17.99 MP） and VI（17.58 MP） scored significantly higher than RU efficacy.The use of self-adhesive cement Rely X Unicem can obtain the bonding strengths to dentin similar to traditional resin cements. Phosphoric acid etching can improve the bonding strengths of the selfadhesive resin cement to enamel, but was negative for dentin.