Proteomics and genetics of dental enamel

The initiation of enamel crystals at the dentino-enamel junction is associated with the expression of dentin sialophosphoprotein (DSPP, a gene normally linked with dentin formation), three 'structural' enamel proteins--amelogenin (AMELX), enamelin (ENAM), and ameloblastin (AMBN)--and a matrix metalloproteinase, enamelysin (MMP20). Enamel formation proceeds with the steady elongation of the enamel crystals at a mineralization front just beneath the ameloblast distal membrane, where these proteins are secreted. As the crystal ribbons lengthen, enamelysin processes the secreted proteins. Some of the cleavage products accumulate in the matrix, others are reabsorbed back into the ameloblast. Once crystal elongation is complete and the enamel layer reaches its final thickness, kallikrein 4 (KLK4) facilitates the breakdown and reabsorption of accumulated enamel matrix proteins. The importance of the extracellular matrix proteins to proper tooth development is best illustrated by the dramatic dental phenotypes observed in the targeted knockouts of enamel matrix genes in mice (Dspp, Amelx, Ambn, Mmp20) and in human kindreds with defined mutations in the genes (DSPP, AMELX, ENAM, MMP20, KLK4) encoding these matrix proteins. However, ablation studies alone cannot give specific mechanistic information on how enamel matrix proteins combine to catalyze the formation of enamel crystals. The best approach for determining the molecular mechanism of dental enamel formation is to reconstitute the matrix and synthesize enamel crystals in vitro. Here, we report refinements to the procedures used to isolate porcine enamel and dentin proteins, recent advances in the characterization of enamel matrix protein posttranslational modifications, and summarize the results of human genetic studies that associate specific mutations in the genes encoding matrix proteins with a range of dental phenotypes.     

CO2 laser-induced zonation in dental enamel: a Raman and IR microspectroscopic study

The gradient of structural alteration and molecular exchange across CO(2) laser-irradiated areas in dental enamel was analyzed by Raman and attenuated total reflectance infrared microspectroscopy. The type and the degree of structural changes in morphologically distinguishable zones within the laser spot vary depending on the laser-irradiation parameters--power (1 and 3 W), treatment time (5 and 10 s), and operational mode (super pulse and continuous wave). Using higher power, irrespective of the operation mode, the enamel tissue ablates and a crater is formed. The prevalent phase at the bottom of the crater is dehydrated O(2) (2-)-bearing apatite, that is, the fundamental framework topology is preserved. Additional nonapatite calcium phosphate phases are located mainly at the slope of the laser crater. No structural transformation of mineral component was detected aside the crater rim, only a CO(3)-CO(2) exchange, which decays with the radial distance. A lower-power laser irradiation slightly roughens the enamel surface and the structural modification of enamel apatite is considerably weaker for continuous wave than for super pulse mode. Prolonged low-power laser treatment results in recrystallization, and thus structural recovering of apatite might be of clinical relevance for enamel surface treatments.     

Correlates of enamel hypoplasia with human dental reduction

Human dental reduction has been manifested in evolutionary and secular trends, but it is not known to what degree each of these complementary processes contributes to changes in tooth size. Enamel hypoplasia is a marker of developmental stress that is often found to be of greater frequency and severity in populations undergoing dental size reduction. In order to test the developmental association of enamel hypoplasia with tooth size, measurements of bucco-lingual and mesio-distal diameters were taken on teeth of 54 black male skulls from southern Africa. Those dentitions that exhibited incisal enamel hypoplasia were significantly reduced in size as compared to those showing no signs of developmental stress. A distinct pattern of reduction emerged: the bucco-lingual diameters of the I¹, I², P³, P⁴, M¹, and M² were significantly reduced, whereas the mesio-distal diameters of only the I² and M² decreased in size. The I² and M² showed the greatest degree of reduction despite the lack of macroscopic enamel hypoplasia on the M². Application of the data to the variety of dental reduction patterns evinced in modern and ancient populations indicates that factors including tooth shape, developmental timing of stress, and genetic determinants of tooth size must be considered in order to partition evolutionary and secular trends in the dentition.     

On the wear mechanism of human dental enamel

The mechanisms and controlling factors in human enamel wear are not fully understood yet. To address this problem, we have used focused ion beam (FIB) milling and field emission scanning electron microscopy (FESEM) to investigate the processes taking place below the wear surface of enamel specimens from in vitro wear tests. These reveal the generation of subsurface cracks during wear of enamel. An analysis of published qualitative and quantitative experimental wear results for enamel as well as for ceramics shows the similarities of the wear processes taking place under similar contact conditions, despite the differences that exist between these two materials. It is shown that, for the considered conditions, fracture under elastic contact is responsible for enamel wear.     

X-ray study and microhardness data of some dental enamel species

X-ray analysis and microhardness of dental enamel of some human and animal species were investigated. Human enamel is composed mainly of hydroxyapatite and fluorapatite together with traces of delta calcium metaphosphate. Prominant apatite phases with a small amount of basic calcium carbonate are also present in the enamel of dog canine teeth. Calcite forms the main crystalline components of rat enamel with some hydroxyapatite. A relatively higher hardness value was detected for enamels of human incisors than for that of rats; the dog enamel was, however, apparently harder than that of the human variety. The hardness values generally decreased with increasing the applied load. The correlation between the phase analysis of the enamel materials and their microhardness values and the dependence of this property on the load applied are discussed.     

Relevance of 2D radiographic texture analysis for the assessment of 3D bone micro-architecture

Although the diagnosis of osteoporosis is mainly based on dual x-ray absorptiometry, it has been shown that trabecular bone micro-architecture is also an important factor in regard to fracture risk. In vivo, techniques based on high-resolution x-ray radiography associated to texture analysis have been proposed to investigate bone micro-architecture, but their relevance for giving pertinent 3D information is unclear. Thirty-three calcaneus and femoral neck bone samples including the cortical shells (diameter: 14 mm, height: 30-40 mm) were imaged using 3D-synchrotron x-ray micro-CT at the ESRF. The 3D reconstructed images with a cubic voxel size of 15 microm were further used for two purposes: (1) quantification of three-dimensional trabecular bone micro-architecture, (2) simulation of realistic x-ray radiographs under different acquisition conditions. The simulated x-ray radiographs were then analyzed using a large variety of texture analysis methods (co-occurrence, spectral density, fractal, morphology, etc.). The range of micro-architecture parameters was in agreement with previous studies and rather large, suggesting that the population was representative. More than 350 texture parameters were tested. A small number of them were selected based on their correlation to micro-architectural morphometric parameters. Using this subset of texture parameters, multiple regression allowed one to predict up to 93% of the variance of micro-architecture parameters using three texture features. 2D texture features predicting 3D micro-architecture parameters other than BV/TV were identified. The methodology proposed for evaluating the relationships between 3D micro-architecture and 2D texture parameters may also be used for optimizing the conditions for radiographic imaging. Further work will include the application of the method to physical radiographs. In the future, this approach could be used in combination with DXA to refine osteoporosis diagnosis.     

Sub-10-micrometer toughening and crack tip toughness of dental enamel

In previous studies, enamel showed indications to occlude small cracks in-vivo and exhibited R-curve behaviors for bigger cracks ex-vivo. This study quantifies the crack tip's toughness (K(I0),K(III0)), the crack's closure stress and the cohesive zone size at the crack tip of enamel and investigates the toughening mechanisms near the crack tip down to the length scale of a single enamel crystallite. The crack-opening-displacement (COD) profile of cracks induced by Vickers indents on mature bovine enamel was studied using atomic force microscopy (AFM). The mode I crack tip toughness K(I0) of cracks along enamel rod boundaries and across enamel rods exhibit a similar range of values: K(I0,Ir)=0.5-1.6MPa?m(0.5) (based on Irwin's 'near-field' solution) and K(I0,cz)=0.8-1.5MPa?m(0.5) (based on the cohesive zone solution of the Dugdale-Muskhelishvili (DM) crack model). The mode III crack tip toughness K(III0,Ir) was computed as 0.02-0.15MPa?m(0.5). The crack-closure stress at the crack tip was computed as 163-770?MPa with a cohesive zone length and width 1.6-10.1μm and 24-44?nm utilizing the cohesive zone solution. Toughening elements were observed under AFM and SEM: crack bridging due to protein ligament and hydroxyapatite fibres (micro- and nanometer scale) as well as microcracks were identified.     

Effects of KrF excimer laser irradiation on human dental enamel

Spectra of human dental enamel was recorded in the 200-400 nm UV region. It showed the weak band at 280 nm which were present in enamel protein. Excimer laser are gas lasers which emit light with photochemical decomposition. The wavelength depends on the 248 nm with krypton-fluoride. The enamel surfaces of extracted human teeth were exposed to KrF excimer laser by an energy density range from 29.6 to 3200 J/cm2. The purpose of this study was to investigate the changes with photo chemical reaction in enamel by light microscopy, SEM and X ray diffraction method. Results of analyses suggested that the observed changes of enamel exposed to this laser were the alpha and beta-tricalcium phosphate (TCP) phase in small amounts. No histological changes were observed in grain boundaries of cross sectioned lased enamel under light microscopy. The SEM examination revealed a roughened surface with bubble formation at 800-3200 J/cm2. SEM of etched enamel surface with 0.1 N HCl after laser irradiation at 400-800 J/cm2 showed the extension along the length of the rods. At 1600-3200 J/cm2, there appeared to be a melting of prism structures, because of conversion of photon energy into thermal energy. These results showed the KrF excimer laser irradiation to dental enamel might be a new type of treatment modality and diagnosis in preventive dentistry.     

Evaluation of enamel dental restoration interface by optical coherence tomography

Evaluation of molar dental restorations on enamel is performed using optical coherence tomography (OCT) with 10 microm resolution. Images of approximately 50 microm failure gaps in the restorations are demonstrated and the OCT images are compared with x-ray and optical microscopy pictures. The results demonstrate the potential of the technique for clinical evaluation of dental restorations.     

不同种齿科修复材料与牙釉质磨损性能的临床评价

背景：冠修复体的严重磨耗会失去咬合功能甚至被磨穿,直接影响修复效果。 目的：评价不同种类常用的牙齿修复材料与牙釉质之间的磨耗性能,寻求与天然牙釉质适应性较好的材料。 方法：由第一作者检索1990-01/2010-02 PubMed数据及万方数据库有关牙齿修补材料及不同修复材料与牙釉质磨损性能关系的相关文献,英文检索词为“dental materials;composite resins; dental enamel;abrasive wear”,中文检索词为“牙釉质;修复材料;磨损;烤瓷;纳米复合体”。根据纳入标准,排除重复性研究,保留20篇文献进行分析。 结果与结论：口腔是一个复杂的电解质环境,酸性饮食,茵斑堆积,细菌新陈代谢作用产生酸性物质,不论是无机酸还是有机酸,都会对金属的耐腐蚀性能产生不良影响。金属材料的化学稳定性好,耐腐蚀性强,与其表面形成的钝化膜有关,而非金属材料的耐腐蚀性则出现很大不同,参差不齐。磨耗性能牙科材料性能优良与否的主要因素,很多实验从牙科材料结构本身、牙齿磨损测量等多方面进行了深入研究,但对牙科材料的磨损机制研究还有待进一步深入。     

High-speed atomic force microscopy of dental enamel dissolution in citric acid

High-speed atomic force microscopy (HS AFM) in 'contact' mode was used to image at video rate the surfaces of both calcium hydroxyapatite samples, often used as artificial dental enamel in such experiments, and polished actual bovine dental enamel in both neutral and acidic aqueous environments. The image in each frame of the video of the sample was a few micrometers square, and the high-speed scan window was panned across the sample in real time to examine larger areas. Conventional AFM images of the same regions of the sample were also recorded before and after high-speed imaging. The ability of HS AFM to follow processes occurring in liquid on the timescale of a few seconds was employed to study the dissolution process of both hydroxyapatite and bovine enamel under acidic conditions. Buffered citric acid at pH values between 3.0 and 4.0 was observed to dissolve the surface layers of these samples. The movies recorded showed rapid dissolution of the bovine enamel in particular, which proceeded until the relatively small amount of acid available had been exhausted. A comparison was made with enamel samples that had been treated in fluoride solution (1 h in 300 ppm NaF, pH 7) prior to addition of the acid; the speed of dissolution for these samples was much less than that of the untreated samples. The HS AFM used an in-house designed and constructed high-speed flexure scan stage employing a push-pull piezo actuator arrangement. The HS AFM is able to follow the large changes in height (on the micrometer scale) that occur during the dissolution process.     

Side effects of a non-peroxide-based home bleaching agent on dental enamel

Changes in the chemistry and structure of enamel due to a non-peroxide-based home bleaching product (Rapid White) were studied in vitro using attenuated total reflectance-infrared spectroscopy, Raman spectroscopy, electron probe microanalysis, flame atomic absorption spectroscopy, and total reflection X-ray fluorescence. The results revealed that the citric-acid-containing gel-like component of the bleaching system substantially impacts on the dental hard tissue. Enamel is affected on several levels: (i) the organic component is removed from superficial and deeper enamel layers and remnants of the bleaching gel are embedded in the emptied voids; (ii) cracks and chemical inhomogeneities with respect to Ca and P occur on the surface; and (iii) within a submicron layer of enamel, the Ca-O bond strength in apatite decreases, thus enhancing calcium leakage from the bleached enamel hard tissue.     

Determination of mineral concentration in dental enamel from X-ray attenuation measurements

The mineral content of dental enamel is commonly measured by X-ray attenuation experiments. Most studies have used contact microradiography in which intensities are measured with photographic film which is convenient and gives high spatial resolution. However photon counting intensity measurements are to be preferred in many experiments (longitudinal and scanning microradiography, and microtomography), as illustrated here, because they have a larger dynamic range and greater sensitivity to small intensity changes. Additionally, the detector and specimen are well separated which allows the pseudo-continuous study of de- and remineralization. The mineral content is often quoted as 95 wt% or 87 vol% hydroxyapatite for permanent human enamel. This determination from attenuation experiments requires accurate values of elemental mass attenuation coefficients and a number of assumptions. The effects of possible choices of these are considered and it is shown that the most important is the density of enamel mineral used in conversion of wt% to vol%. If the density is taken as 2.99 g cm(-3), as recently suggested (J.C. Elliott, Dental Enamel, Ciba Foundation Symposium 205, Wiley, Chichester, pp. 54-72, 1997), instead of 3.15 g cm(-3) as for hydroxyapatite, the calculated vol% is approximately 93 instead of approximately 87.     

Impedance of dental enamel membranes as a predictor for their permeability

The permeability of dental enamel membranes can be estimated by means of diffusion-measurements with radio-isotopes. Because of the low intrinsic permeability of these membranes, determination via this route generally takes two weeks. In this paper a much faster electrochemical method is presented, in which the real and imaginary part of the membrane impedance are measured at discrete intervals in the frequency range 10 Hz–300 kHz. From the parameters that describe these electrochemical data, some were found to correlate closely with the permeability of enamel as determined with radio-isotopes on the same enamel sample. From these results it was concluded that especiallyv _max, the frequency where the imaginary part of the membrane impedance is maximal, and Z₁ at 100 Hz, the imaginary part of the membrane impedance at 100 Hz, are of practical value as predictor for the permeability of enamel.     

Effect of Nd:YAG laser irradiation on human dental enamel

The crystallographic alterations of the hydroxyapatite under the laser irradiation were evaluated by the x-ray diffraction pattern analysis and scanning electron microscopic observation. Recrystallization may occur in fused and resolidified dental enamel by the pulsed Nd: YAG laser irradiation. Two different grades of energy densities such as 400 pulses and 800 pulses were given to the powdered enamel. The irradiation was performed at a peak power of 500W with a pulse width of 10 msec.; the average output of 10W, spot size of 3mm and two pulses were given in every second. After the 800 pulses of Nd:YAG laser irradiation to the human dental enamel the x-ray diffraction pattern demonstrated both alpha-tricalcium phosphate and hydroxyapatite peaks. With the lower level of energy at 400 pulses, no significant differences were seen in the diffraction patterns between lased and unlased enamel. In scanning electron microscopic findings, there were no significant changes between lased and unlased enamel. When the unlased and lased enamel were exposed to acid solution, unlased enamel showed a honeycomb pattern, while the lased enamel showed preferentially removed prism core material.     

Demineralization and ion binding action of polycarboxylate cement liquid on human dental enamel

The demineralization and ion binding effects on dental tissues due to poly(acrylic) acid attack under different dilution conditions of the latter are studied by scanning electron microscope (SEM) and infrared (IR) spectroscopy. The results indicate that in addition to the general demineralization effects such as preferential prism core attack, prism periphery attack, and protruding prism rods, competing ion binding effects also occur. Using optimum dilution conditions, the microstructural effects of demineralization and ion binding are shown and the effects are related to IR spectral observations.     

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.     

Light scattering properties of natural and artificially demineralized dental enamel at 1310 nm

A fundamental understanding of how near-IR light propagates through sound and carious dental hard tissues is essential for the development of clinically useful optical diagnostic systems, since image contrast is based on changes in the optical properties of these tissues on demineralization. During the caries (decay) process, micropores are formed in the lesion due to partial dissolution of the individual mineral crystals. Such small pores behave as scattering centers, strongly scattering visible and near-IR light. The optical properties of enamel can be quantitatively described by the absorption and scattering coefficients, and the scattering phase function. Our aim is to measure the optical scattering behavior of natural and artificial enamel caries. Near-IR attenuation measurements and angular-resolved goniometer measurements coupled with Monte Carlo simulations are used to determine changes in the scattering coefficient and the scattering anisotropy on demineralization at 1310 nm. An ultra-high resolution digital microradiography system is used to quantify the lesion severity by measurement of the relative mineral loss for comparison with optical scattering measurements. The scattering coefficient increases exponentially with increasing mineral loss. Natural and artificial demineralization increases the scattering coefficient more than two orders of magnitude at 1310 nm, and the scattering is highly forward directed.     

The orientation of prisms in the dental enamel of human permanent teeth

Enamel of human permanent teeth was sectioned and ground with 2 planes perpendicular to each other extending centrifugally from the dentino-enamel-junction to the crown surface. Prisms were made visible by acid etching before evaluation under the SEM. In the vicinity of the dentino-enamel-junction more prisms were found to be cut longitudinally, while close to the crown surface more prisms were cut transversely. In the perpendicularly ground plane the corresponding prisms were seen to deviate the more from the centrifugal orientation the more close they came to the crown surface. In a geometric model the angle under which the prisms deviate from the centrifugal orientation was calculated in dependence from the distance to the dentino-enamel-junction. The results correspond with the SEM-findings. We conclude that--since the prism diameter is today known to be constant--the form of the enamel mantle is created by a specific orientation of the prisms: They stand perpendicular at the dentino-enamel-junction, and the farther away they run towards the periphery the more they deviate from the perpendicular path. By this increasingly oblique orientation of the prisms the volume increment of the enamel mantle is created. The maximal angle of deviation is found morphologically and mathematically between 60 degrees-70 degrees at the crown surface. This arrangement of the prisms demonstrated by us is now seen to be the reason for erroneous assumptions about an increase of the prism diameter when ground sections were used. Because the prisms are oriented more and more oblique towards the periphery, correspondingly larger effective diameters must be produced while making ground sections tangential to the crown surface.