Enamel Hydroxyapatite Research Articles

Human cementum tumor cells have different features from human osteoblastic cells in vitro.

Cells obtained from human cementoblastoma and alveolar bone were isolated and cultured. Initial and late stages of mineralization were assessed by using atomic force microscopy, scanning electron microscopy and X-ray microanalysis. In cultures of cementoblastoma-derived cells the initial stages of mineralization showed well-defined spherical-shaped structures, while the osteoblastic cells showed plaque-like deposits. These morphological patterns of mineral deposition could serve as nucleation centers for hydroxyapatite crystals. Late stages of mineralization at 28 and 35 d maintained those morphological differences established in initial cultures. The material deposited by cementoblastoma and osteoblastic cells, analyzed by EDX spectra, revealed similar Ca/P ratios for both cell types. These values were similar to those reported for hydroxyapatite in enamel and bone. Alkaline phosphatase specific activity (AlP), of osteoblastic cells at 3, 7 and 11 d, showed an increase of 27.9, 50.9 and 37.0% (p < 0.001), respectively. However, at 15 and 19 d there was an increase of AlP activity of cementoblastoma cells by 39.4 and 34.5% over osteoblastic cells (p < 0.001). Immunostaining of cementoblastoma and osteoblastic cells using a specific mAb against a cementum-derived attachment protein revealed strong immunostaining of cementoblastoma cells which was localized to the cell membrane and fibril-like structures (96.2 +/- 1.3). A few osteoblastic cells also stained weakly with the anti-CAP mAb (6.4 +/- 0.6). Sections of decalcified paraffin embedded cementoblastoma specimens, when immunostained with anti-CAP mAb, showed strong immunostaining of the cells surrounding the regular and irregularly-shaped calcified masses of the tumor. Putative cementocytes also stained positively. Immunostaining with a polyclonal antibody against osteopontin strongly stained the osteoblastic cells (89.0 +/- 3.6). Cementoblastoma cells showed weaker staining (54.2 +/- 2.4). The results suggest that cementoblastoma cells could be a major source of specific cementum proteins. These cells could provide the opportunity to elucidate the regulation of the cementogenesis process.

Human cementum tumor cells have different features from human osteoblastic cells in vitro

Cells obtained from human cementoblastoma and alveolar bone were isolated and cultured. Initial and late stages of mineralization were assessed by using atomic force microscopy, scanning electron microscopy and X‐ray microanalysis. In cultures of cementoblastoma‐derived cells the initial stages of mineralization showed well‐defined spherical‐shaped structures, while the osteoblastic cells showed plaque‐like deposits. These morphological patterns of mineral deposition could serve as nucleation centers for hydroxyapatite crystals. Late stages of mineralization at 28 and 35 d maintained thosemorphological differences established in initial cultures. The material deposited by cemetoblastoma and osteoblastic cells, analyzed by EDX spectra, revealed similar Ca/P ratios for both cell types. These values were similar to those reported for hydroxyapatite in enamel and bone. Alkaline phosphatase specific activity (Alp), of osteoblastic cells at 3, 7 and 11 d, showed an increase of 27.9, 50.9 and 37.0% (p&lt;0.001), respectively. However, at 15 and 19 d there was an increase of A1P activity of cementoblastoma cells by 39.4 and 34.5% over osteoblastic cells (p &lt;0.001). Immunostaining of cementoblastorna and osteoblastic cells using a specific mAb against a cementum‐derived attachment protein revealed strong immunostaining of cementoblastoma cells which was localized to the cell membrane and fibril‐like structures (96.2±1.3). A few osteoblastic cells also stained weakly with the anti‐CAP mAb (6.4 ±0.6). Sections of decalcified paraffin embedded cementoblastoma specimens, when immunostained with anti‐CAP mAb, showed strong immunostaining of the cells surrounding the regular and irregularly‐shaped calcified masses of the tumor. Putative cementocytes also stained positively. Immunostaining with a polyclonal antibody against osteopontin strongly stained the osteoblastic cells (89.0±3.6). Cementoblastoma cells showed weaker staining (54.252.4). The results suggest that cementoblastoma cells could be a major source of specific cementum proteins. These cells could provide the opportunity to elucidate the regulation of the cementogenesis process.

Protein-to-Protein Interactions: Criteria Defining the Assembly of the Enamel Organic Matrix

Enamel crystallites form in a protein matrix located proximal to the ameloblast cell layer. This unique organic extracellular matrix is constructed from structural protein components biosynthesized and secreted by ameloblasts. To date, three distinct classes of enamel matrix proteins have been cloned. These are the amelogenins, tuftelin, and ameloblastin, with recent data implicating ameloblastin gene expression during cementogenesis. The organic enamel extracellular matrix undergoes assembly to provide a three-dimensional array of protein domains that carry out the physiologic function of guiding enamel hydroxyapatite crystallite formation. Using the yeast two-hybrid system, we have surveyed these three known enamel gene products for their ability to direct self-assembly. We measured the capacity of the enamel gene products to direct protein-to-protein interactions, a characteristic of enamel proteins predicated to be required for self-assembly. We provide additional evidence for the self-assembly nature of amelogenin and tuftelin. Ameloblastin self-assembly could not be demonstrated, nor were protein-to-protein interactions observed between ameloblastin and either amelogenin or tuftelin. Within the limits of the yeast two-hybrid assay, these findings constrain the emerging model of enamel matrix assembly by helping to define the limits of enamel matrix protein-protein interactions that are believed to guide enamel mineral crystallite formation.

Retrospective Accident Dosimetry Using Environmental Materials

Abstract Retrospective dosimetry of radiation accidents can be accomplished with the aid of environmental materials which were exposed during the time of the accident. The primary requirements for such dosemeters include: (1) the absence of a saturating natural dose accumulated over geological time periods, (2) the ability to retain a record of the accident exposure with negligible fading over the time interval between exposure and measurement and (3) the ability to determine the cumulative dose from natural radiation sources. Natural dosemeters often meeting these requirements are the quartz inclusions found in fired ceramics such as bricks, tiles and porcelain plumbing fixtures. The firing during manufacture erases the prior geological dose. The total cumulative dose (DT) accrued from natural sources as well as the accidental dose is measured using thermoluminescence or other dosimetry techniques. The cumulative natural dose since firing (DN) can de determined with a knowledge of the age of the material (time since firing) together with measurements of dose rate due to internal as well as external sources. The accidental dose is derived by subtracting the natural from the total accrued dose (DT-DN). In cases of high accidental exposures or low natural doses due to the selection of young samples, the natural dose can be estimated or neglected. Three dosimetry techniques, the pre-dose TL technique, the 'high temperature' TL technique (quartz inclusion or fine grain) and more recently the optically stimulated luminescence technique may be applied to quartz grains from the same samples to provide estimates of accrued dose which are largely independent of one another. A non-destructive technique, electron paramagnetic resonance spectroscopy (EPR) may also be applied to these materials in situations of moderately high exposure. An additional advantage of EPR is that it may be applied to biological materials, most notably the hydroxyapatite of tooth enamel. This paper briefly reviews past uses of dosimetry techniques in accident situations, discusses the types of information which may be obtained with their use and the basic methodologies of some of the procedures.

Laser induced molar tooth pulp chamber temperature changes.

Temperature changes in enamel tissue and the pulp chamber under the influence of a CO2 laser were measured by direct methods in vitro. X-ray diffraction analysis revealed alpha-Ca3(PO4), the high-temperature modification of enamel hydroxyapatite, thus indicating that the enamel melting temperature was above 1,000 degrees C in the interaction area of laser (continuous wave, 15 s exposure time, 1 mm spot size) and tissue. Powers of 0.5 and 1 W (continuous wave), 1.5 mm spot size, and 10 s exposure time vaporize and carbonize dentin tissue at the cavity bottom of class I preparation molars. The observed temperature rise of 4 degrees C indicates that thermal injury to the pulp tissue does not occur.

ESR dating of tooth enamel: comparison with [formula omitted] speleothem dates at La Chaise-de-Vouthon (Charente), France

One way to assess a new dating method's reliability is by comparing its results with those from well established, independent techniques. A controlled test of the electron spin resonance (ESR) dating method as it is currently being applied to teeth was attempted for the time range 100–250 ka, beyond that of 14C, at the archaeological site of La Chaise-de-Vouthon (Charente, France). Although absent in modern enamel, a single ESR signal with g = 2.0018 in fossil tooth enamel hydroxyapatite increases in amplitude with increasing irradiation doses. ESR ages are derived from the ratio of the AD, the radiation dose needed to produce the observed ESR signal, relative to the natural, environmental dose rate (ED) experienced by the tooth after deposition. Since the age depends on the uranium (U) uptake history assumed, three ages are calculated assuming: (1) early U uptake (EU); (2) continuous (linear) uptake (LU); (3) recent uptake (RU). Generally, the LU age agrees best with known ages determined by other methods, although the RU model is better for some teeth. ESR dating assumes that the fossil has not suffered recrystallization or significant diagenetic alteration. In the preliminary test, three teeth were dated. In Bourgeois-Delaunay, a bovid molar associated with Palaeolithic artefacts was collected from layers dated at 101 ± 12 to 114 ± 7 ka by 230Th 234U dating of the over- and underlying stalagmitic floors. From Suard, two Equus teeth were collected from beneath a stalagmitic floor dating 112 ± 12 ka. ESR dating teeth significantly underestimated the true age for the teeth: the mean ESR ages range from 37 to 94 ka with standard errors of 2–6 ka, and good replicability. Although more teeth at La Chaise need to be tested to ascertain that the underestimation does not result from random variation commonly seen among teeth within one unit, the consistent underestimation suggests a fault in one of the assumptions underlying the dating method. The most obvious source of error lies in the difficulty in modelling the external γ dose. Only U leaching, not incorrectly modelled U uptake, would cause the underestimation. Diagenetic alteration may also cause anomalous fading, thermal instability, variation in k, or ESR signal suppression. More study into the effects of diagenesis alteration on enamel ESR signals is needed, as is a reevaluation of the mean signal life and α efficiency for several more enamel samples.

Effect of Pore Topology on the Remineralization Rate of Surface-Softened Enamel and Synthetic Hydroxyapatite: An Experimental Comparison and Mathematical Model

Experiments with surface-softened synthetic hydroxyapatite show similar remineralization rates as surface-softened human enamel. The mineral gain with respect to time relates to parameters governing the precipitation of mineral in the specimens. A mathematical model regarding the mineral precipitation on the pore surface is fitted to the experimental values. We obtained comparable precipitation rates for apatite and tooth enamel. The geometry of the pores, however, proved to be different: a mostly tube-like pore structure must be attributed to demineralized enamel, while the synthetic hydroxyapatite specimens remineralize like a ramified, crack-like microstructure of pores.

Development of an automated experimental setup for the study of ionic-exchange kinetics. Application to the ionic adsorption, equilibrium attainment and dissolution of apatite compounds

An ion-selective electrode and microcomputer-based experimental setup for the study of ionic-exchange kinetics between a powdered solid and the solution is described. The equipment is composed of easily available commercial devices and a data acquisition and regularization computer program is presented. The system, especially developed to investigate the ionic adsorption, equilibrium attainment and dissolution of hard mineralized tissues, provides good reliable results by taking into account the volume changes of the reacting solution and the electrode behaviour under different experimental conditions, and by avoiding carbonation of the solution. A second computer program, using the regularized data and the experimental parameters, calculates the quantities of protons consumed and calcium released in the case of equilibrium attainment and dissolution of apatite-like compounds. Finally, typical examples of ion-exchange and dissolution kinetics under constant pH of enamel and synthetic hydroxyapatite are examined.

Crystal Morphology and Decalcification Patterns Compared in Rat and Human Enamel and Synthetic Hydroxyapatite

The purpose of this investigation was to compare morphology and dissolution patterns by ultrastructural examination of rat and human enamel crystals as well as synthetic apatite crystals. Mature enamel crystals were of particular interest, since crystal maturation appears to be inhibited in amelogenesis imperfecta. Specimens were isolated from developing and mature rat incisor enamel. Rat enamel, mature human enamel, and synthetic apatite were thin-sectioned without decalcification and examined by transmission electron microscopy. Some sections were exposed to acid, and selected synthetic apatite sections were further treated for removal of embedding plastic, followed by vacuum-shadow-coating with carbon. Results showed that cross-sections of rat, human, and synthetic crystals had a distortion in the flattened hexagonal outline in regions where the growth of one crystal impinged on another. Crystal dissolution occurred preferentially along the c-axis, producing a central defect or hole in the crystals. Preliminary studies with weak acid on mature human enamel indicate that the relatively soluble crystal core is quickly dissolved, while the outer shell remains intact over a much longer period of time. In the mature rat and human enamel, this crystal hole formation had a consistent dimension of approximately 10-nm thickness. The crystal hole dimension was the same size as crystals that are formed during the early secretory phase in rat amelogenesis. Acid-treated synthetic apatite also showed dissolution of the crystal core along the c-axis, but dimensions of the hole were not consistent.(ABSTRACT TRUNCATED AT 250 WORDS)

ESR dating of tooth enamel from prehistoric archaeological sites

Electron spin resonance (ESR) can be used to date hydroxyapatite of tooth enamel, a common component of prehistoric archaeological sites. Dates, ranging from 10 ka to ∼ 3Ma, are calculated from the accumulated radiation dose, A, and the dose rate, D, which varies with time as a result of uptake of U and growth of U-daughters to secular equilibrium.

Direct Structure Imaging In Hydroxyapatite

Human tooth enamel is composed 96% by weight of carbonated hydroxyapatite (HAP). It Js believed that the carbonate ions substitute for the phosphate ions in HAP, and it is expected that the carbonate will have an effect on the rate of dissolution of HAP during the carious process in human tooth enamel. In the present study high resolution electron microscope images of synthetic stoichiometric HAP were obtained to explore the possibility of obtaining direct structure images in HAP, and to provide a standard to which human enamel HAP could be compared. Bres et al have pointed out that structure images of HAP may provide a means of studying the substitution by carbonate of phosphate or other ions in biological HAP. The specimens were examined with a top-entry JEOL 4000EX HREM fitted with a Cs = 1.0mm objective pole-piece at 400kV. This 4000EX has demonstrated a structure resolution limit of better than 1.8 Å.

Structure of crystals in dental enamel and synthetic hydroxyapatite

Biological apatites in dental enamel, dentine and bone closely resemble, in chemical composition, the mineral hydroxyapatite with the unit cell Ca10(PO4)6(OH)2. Substitutions occur frequently, e.g. fluoride in place of hydroxy groups, carbonate for phosphate, and with other elements such as magnesium, zinc, strontium, and lead instead of calcium. The substituents are known to influence the response of the material to attack by dilute organic acids and are, therefore, of importance in the process of dental caries.The sub-lattice resolution available with some modern electron microscopes makes it feasible to attempt detection of the effect of substituted groups on the structure of the hydroxyapatite crystals. As a starting point in such a venture we have obtained high-resolution images of apatite crystals and used computer image calculation techniques to interpret the micrographs at near-atomic resolution.

Properties of dissociatively extracted fetal tooth matrix proteins. I. Principal molecular species in developing bovine enamel.

A sequential dissociative extraction scheme is described in which tooth matrix proteins are extracted first in 4 M guanidine HCl, pH 7.4, and then in 4 M guanidine HCl, 0.5 M EDTA, pH 7.4, both with protease inhibitors present. The latter step dissolves the mineralized portion of the tissue and extracts noncollagenous proteins closely associated with hydroxyapatite crystallites in the mineralized matrix. In fetal bovine enamel, the initial dissociative extraction step completely removes proline-rich amelogenins from the tissue without dissolving the enamel apatite. The amelogenin proteins consist of several species on polyacrylamide gel electrophoresis with sodium dodecyl sulfate, but display anomalous migration behavior relative to conventional marker proteins in this technique. Subsequent extraction of fetal bovine enamel with guanidine HCl/EDTA removes matrix enamelins, acidic glycoproteins that are tightly bound to the enamel hydroxyapatite. This latter fetal protein type has not been isolated previously. The enamelins are adsorbed strongly by DEAE-cellulose in 7 M urea and totally adsorb to synthetic apatite, even in 4 M guanidine HCl. The enamelins display normal behavior on polyacrylamide gels and stain positively for sialic acid/phosphate and carbohydrate. With advancing tooth maturation, amelogenins disappear while enamelins are conserved. Gel filtration chromatography in 4 M guanidine HCl showed amelogenin components at apparent molecular weights of approximately 25,000, 15,000, 9,500, 7,500, and 6,000, while the enamelins eluted at Mr positions of approximately 72,000, 56,000, 42,000, 30,000, 21,000, 13,000, and 8,000. The gel filtration data showed a clear shift in molecular size population from higher to lower components for both amelogenins and enamelins with progressive enamel maturation.

The kinetics of inorganic phosphate in human dental plaque and saliva

The kinetics of inorganic phosphate (P i), Na and K in plaque fluid and changes in the composition of the residual 24 h human dental plaque was studied following rinsing with water, 20 per cent sucrose, 0.12 M and 0.5 M phosphate. Rinsing with sucrose solution reduced plaque P i to a greater extent than water, and the effect was demonstrable 10 min later. The main effect of both on plaque P i was due to the exposure of plaque to a P i-free solution. Rinsing with P i elevated plaque fluid P i but the effect was short-lived and not detectable 15 min later irrespective of the concentration used. Changes in the Na and K concentrations in plaque fluid after sucrose could not be related to the effects of bacterial metabolism. Phosphate added to sucrose and ingested simultaneously was cleared from the saliva at a time when substantial amounts of carbohydrate remained. The effect of sucrose in lowering plaque fluid P i was prevented by 0.12 M P i introduced separately after the sucrose and was reversed by 0.5 M P i. It was concluded that the local effect of raising plaque P i would not contribute significantly to inhibition of acid production, desorption of protein from enamel hydroxyapatite or buffering and that when P i was introduced into plaque separately from, and after, sucrose the elevated plaque fluid P i could exert a short-lived local effect, by mass action, on the dissolution and remineralization of enamel.

Laser-induced effects on tooth structure. VI. X-ray diffraction study of dental enamel exposed to a CO2 laser.

The aim of the study was to investigate crystallographic changes in lased dentine. With the aid of the X-ray diffraction method it was shown that recrystallization occurred in dentine due to laser irradiation. Simultaneously, growth in the crystal size of the crystallites was observed, and dentine of a low order of crystallinity changed structurally in such a way that it came to closely resemble the crystalline structure of the hydroxyapatite of normal enamel.

The crystal structure of hydroxyapatite in dental enamel as seen with the electron microscope

Thin sections of rat incisor enamel were studied with the electron microscope. Repeating patterns of minute striations, representing the resolution of sets of lattice planes, were observed in images of individual enamel crystals. In cross-sectioned crystals, fringe patterns with 8.2 repeat period representing the 100 planes of the hydroxyapatite lattice occurred. The hexagonal packing of the unit cells in this projection could be demonstrated. Crystals oriented with their c axis in the plane of the section showed, in addition to the 100 planes, a transverse striation pattern with a periodicity of 3.4 , which was considered to represent the 002 planes. Additional spacings were observed, confirming the interpretation of the orientation of the crystals. With this, the molecular arrangement of hydroxyapatite in dental enamel has been demonstrated visually not only in a projection normal to, but also parallel to, the long axis of the crystals.