Crystallographic structure of human tooth enamel by electron microscopy and x-ray diffraction: hexagonal or monoclinic?

Recently reports on the major stability of the monoclinic phase of hydroxyapatite compared with the hexagonal phase have established it as the most observable structure of hydroxyapatite in natural materials, such as hard tissues. In this work, the structural and crystallographic analysis of the inorganic component of sound human tooth enamel was done by transmission electron microscopy, electron diffraction and X-ray diffraction techniques. The results indicated that its unit cell is hexagonal not monoclinic.

Surface structure study of biological calcium phosphate apatite crystals from human tooth enamel.

A surface-structure study of human tooth enamel crystals has been carried out by high-resolution electron microscopy (HREM). The surfaces of several crystals have been examined and the surfaces of a single crystal are described here. The observations made on this crystal are similar to the observations made on the other crystals, although the difference of morphology in the crystals observed indicates that the growth control by matrix proteins takes only place on the [011 macro 0] surfaces of the crystals. The crystal described here is oriented along the [112 macro 0] direction, and the following surfaces have been analyzed: (11 macro 00), (011 macro 1), (01 macro 11) and (0001). A subsurface reconstruction is observed just below the surface of the crystal not bonded to the matrix and lying above the supporting film. Observation of the matrix surrounding the crystal shows the existence of poorly crystalline phases with a structure close to that of hydroxyapatite (OHAP). Finally, a comparison of the images of the (011 macro 0) surface with computer-simulated images calculated for several models of the OHAP surface structure shows that the surface itself is stoichiometric and contains both calcium and phosphate groups. The knowledge of the binding sites of proteins on biomineral crystals such as the ones found in human tooth enamel is of prime importance for the understanding of their growth process. In this study, the first structure determination of the surface of human tooth enamel crystals is presented.

CpG oligodeoxynucleotides with hepatitis B surface antigen (HBsAg) for vaccination in HBsAg-transgenic mice.

DNA motifs containing unmethylated CpG dinucleotides within the context of certain flanking sequences enhance both innate and antigen-specific immune responses, due in part to the enhanced production of Th1-type cytokines. Here we explored the ability of CpG-containing oligodeoxynucleotides combined with recombinant hepatitis B surface antigen (HBsAg) to induce Th1 responses in mice that are transgenic for this antigen and that represent a model for asymptomatic hepatitis B virus chronic carriers. This was compared to hepatitis B virus-specific DNA-mediated immunization, which we have previously shown to induce the clearance of the transgene expression product and the down-regulation of hepatitis B virus mRNA in this transgenic mouse lineage. In control nontransgenic C57BL/6 mice, three immunizations with HBsAg and CpG triggered the production of anti-HBs antibodies and of HBs-specific T cells that secrete gamma interferon but do not display any HBsAg-specific cytotoxic activity. In the HBsAg-transgenic mice, immunization with HBsAg and CpG oligodeoxynucleotides, but not with CpG alone, induced the clearance of HBsAg circulating in the sera, with a concomitant appearance of specific antibodies, and was able to regulate the hepatitis B virus mRNA constitutively expressed in the liver. Finally, adoptive transfer experiments with CD8(+) T cells primed in C57BL/6 mice with HBsAg and CpG oligodeoxynucleotide-based immunization show that these cells were able to partially control transgene expression in the liver and to clear the HBsAg from the sera of recipient transgenic mice without an antibody requirement. CpG oligodeoxynucleotides motifs combined with HBsAg could therefore represent a potential therapeutic approach with which to treat chronically infected patients.

Structure and substitutions in fluorapatite.

Fluorapatite, Ca10(PO4)6F2. is a widely spread form of calcium phosphate present particularly in biological material. Human hard tissues contain crystals structurally related to apatite. Fluoride can be found in various natural sources and is also used for its beneficial action in caries prevention. Fluorapatite belongs to the spatial group P6(3/m) (C(6h)2) and consists of 3 ions: F-, Ca2+, PO4(3-). In the present paper, we have carried out a crystallographic study of the fluorapatite structure and of the changes induced by the substitutions. The fluorapatite structure and the presence of a large number of ionic bonds make fluorapatite a very suitable host for many substituents, some of them harmless for the human organism, some not. According to the substitution site, we can describe four types of substitution. The F- substitution, also called Type A substitution, is the main one, and the best known. Only the Ca2+ substitution implies changes in the crystal structure. However, some questions remain, in particular for the PO4(3-) substitution, which is the main substitution present in the biological calcium phosphates.

MicroRaman spectral study of the PO4 and CO3 vibrational modes in synthetic and biological apatites.

The carbonate and phosphate vibrational modes of different synthetic and biological carbonated apatites were investigated by Raman microspectroscopy, and compared with those of hydroxyapatite. The nu1 phosphate band at 960 cm-1 shifts slightly due to carbonate substitution in both A and B sites. The spectrum of type A carbonated apatite exhibits two nu1 PO43- bands at 947 and 957 cm-1. No significant change was observed in the nu2 and nu4 phosphate mode regions in any carbonated samples. The nu3 PO43- region seems to be more affected by carbonation: two main bands were observed, as in the hydroxyapatite spectrum, but at lower wave numbers. The phosphate spectra of all biominerals apatite were consistent with type AB carbonated apatite. In the enamel spectrum, bands were observed at 3513 and at 3573 cm-1 presumably due to two different hydroxyl environments. Two different bands due to the carbonate nu1 mode were identified depending on the carbonate substitution site A or B, at 1107 and 1070 cm-1, respectively. Our results, compared with the infrared data already reported, suggest that even low levels of carbonate substitution induce modifications of the hydroxyapatite spectrum. Increasing substitution ratios, however, do not bring about any further alteration. The spectra of dentine and bone showed a strong similarity at a micrometric level. This study demonstrates the existence of acidic phosphate, observable by Raman microspectrometry, in mature biominerals. The HPO42- and CO32- contents increase from enamel to dentine and bone, however, these two phenomena do not seem to be correlated.

[Long-term ultrasonographic evaluation of teflon paste compression in children endoscopically treated for vesicoureteral reflux. Preliminary report]. / Odlegla ultrasonograficzna ocena skupiska pasty teflonowej u dzieci leczonych endoskopowo z powodu odplywu pecherzowo-moczowodowego. Doniesienie wstepne.

UNLABELLED: The endoscopic treatment of vesicoureteral reflux with transurethral injection of teflon into submucosal part of ureter was performed for the first-time in 1981. Unfortunately this method of treatment was not accepted because of the teflon migration (proved in experimental therapy) into different organs (lungs, brain, lymphatic nodules). Defining the quantity changes of teflon in children treated with this method in long-term follow-up (minimum 8 years). The ultrasonography was performed in 20 cases of female children with diagnosed vesicoureteral reflux and treated in 1987-1990 by injection of teflon (in age from 1 to 10). There were 33 ureters treated. The vertical and transversal sizes and the area of teflon were defined and compared with ultrasonographic investigation performed early after injection. In 14 ureters (43%) the sizes of teflon mass in submucosal part of the ureter were the same as just after injection. In 12 ureters (36%) the mass was considerably lower and in 7 cases (21%) there was no teflon in the injection place. The changes of teflon mass quantity were noticeable in 57%. CONCLUSIONS: The reduction or loss of teflon mass injected into submucosal part of ureter in children treated endoscopically for vesicoureteral reflux was demonstrated in high percentage of cases. There is a possibility that teflon was excreted into bladder through mucosa perforated by a needle during injection or because of later perforation of mucosa. This hypothesis is not so probable--early postoperative ultrasound controls showed right quantity of teflon mass. There is no proof that the loss of mass is caused by migration. This situation has to be taken into consideration but the appearance of symptoms is rather not probable (quantity of injected mass varies 0.1-1 ml).

Basic fibroblast growth factor adsorption and release properties of calcium phosphate.

Basic fibroblast growth factor (bFGF) is well known as a potent angiogenic factor playing a crucial role in wound-healing processes. Apatitic substrates such as hydroxyapatite and carbonated apatite (CA) could be potential carriers of growth factors because of their physicochemical similarities to bone mineral. These materials have been compared for their bFGF adsorption and release properties. The adsorption of the growth factor was higher on carbonated apatite than on hydroxyapatite, probably owing to environments with labile nonapatitic CO3(2-) and HPO4(2-) groups, along with a higher specific surface area which gives the CA a higher surface reactivity. These environments can be exchanged very rapidly, leading to the release of bFGF. The controlled release of adsorbed growth factor from carbonated apatite could provide means of improving bone healing in the future.

Infrared and Raman microspectrometry study of fluor-fluor-hydroxy and hydroxy-apatite powders.

Visible Raman and infrared microspectrometry studies performed on fluorapatite and hydroxyapatite powders have shown similar results. Small modifications of the nu2 and nu4 PO(3-)4 tetrahedra bending modes are observed. A small frequency shift of the nu1 mode and modifications on the nu3 mode region accompanied with a simplification of the hydroxyapatite and fluorapatite respective spectra from seven to four bands were observed. A broad and weak band which could be attributed to the Ca-F bond is detected at 311 cm(-1) on the Raman fluorapatite spectra. The phosphate vibration modes are little disturbed by fluoride substitution. This could indicate that phosphate groups interact strongly between themselves and weakly with substituted atoms (i.e. hydroxyle and fluoride atoms). Whatever crystallographic model is considered, the number of bands observed is always lower than the number of calculated ones, even for hydroxyapatite, whose symmetry is lower than that of fluorapatite.

Study of interface phenomena between bone and titanium and alumina surfaces in the case of monolithic and composite dental implants.

The interface between mandibular bone and dental implants was examined with the in vivo dog model. Implant/bone interfaces were investigated for three types of materials: Ti-30 wt% Ta/Al2O3, titanium and Al2O3 using microscopy techniques covering a large magnification range: scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray analysis and Auger spectroscopy. During the interaction of the Al2O3 ceramic with bone, an interfacial layer about 15 microm thick is formed. The same phenomenon was observed at the titanium bone interface, where the thickness of the layer was about 10 microm. In all cases, interface layers were sharp with well-defined borders between bone tissue and implant materials. No calcification took place inside the interface layer. A chemical analysis performed on this layer shows the presence of titanium, calcium and phosphorus in the case of titanium implants, and aluminium, calcium and phosphorus in the case of alumina implants. A rapid decrease in metal composition with increasing distances from the implant surface is correlated to a slow increase in calcium and phosphorus in the direction of the bone. Direct contact between implant and bone was observed. No biocorrosive effects were detected at the Ti-30 wt% Ta/Al2O3 metal-ceramic interface.

Observation of the loss of the hydroxyapatite sixfold symmetry in a human fetal tooth enamel crystal.

A deviation from the hydroxyapatite hexagonal symmetry of a human tooth enamel crystal observed by high-resolution electron microscopy is reported. This symmetry deviation is characterized by: (1) 'preferential' planes that can be indexed as (100) with an intensity that differs from the (300) and the other (100) hexagonal equivalent planes; and (2) streaking of higher order reflections in the optical diffractogram of the image of the crystal. Computer simulations show that similar 'preferential' planes can also be observed at specific crystal tilt angles (and/or beam tilt and/or objective aperture misalignment) and at crystal thickness/microscope defocus values in images of hydroxyapatite crystals observed along the [0001] or [2243] zone axes. The streaking of higher order reflections in the optical diffractogram is related to a deformation of the crystal itself and does indeed show a symmetry deviation of the crystal under observation.

Space-group determination of human tooth-enamel crystals.

In the present work, we have determined the space group of human tooth-enamel crystals using--for the first time for a biological crystal--convergent beam electron diffraction (CBED). The symmetries observed in the different patterns we have obtained lead us to the P6(3)/m hydroxyapatite space group. Disorder, most likely situated in the columns formed by the hydroxyl ions of the crystals, is suggested as a cause of weak intensity in the otherwise forbidden 000l (l odd) reflections and low visibility of first-order Laue zone (FOLZ) reflections in the CBED pattern from crystals oriented along the [0001] zone axis. A monoclinic phase was not observed.

Compositional variations in apatites with respect to preferential ionic extraction.

Detection of ionic losses from the apatitic structure (Ca10(PO4)6(OH)2) by high-resolution electron microscopy was investigated theoretically. Linear image analysis showed the need for an objective aperture of at least 3.7 nm-1 to visualize four different coordinates (CaII, OH-, P and mid-point between CaII-P bond). High-resolution image analysis and plotting of OH- column intensity against specimen thickness showed an inverse proportionality between composition and OH- image intensities for very thin specimens (less than 2 nm). Image intensity variation would be detectable experimentally, but the preparation of such thin specimens by ultramicrotomy is impossible.

Adsorption/desorption of human serum albumin on hydroxyapatite: a critical analysis of the Langmuir model.

We studied the adsorption of human albumin onto synthetic hydroxyapatite, using a radiotracer technique and a special flow cell. Adsorption was studied under various conditions corresponding to different thermodynamic paths. It appears that (i) as is the usual case, the isotherms obtained within a short time range (a few hours) do not correspond to a true equilibrium situation; (ii) when the adsorption process is followed for longer times, which is necessary at low bulk concentrations, one always reaches the plateau surface adsorption; (iii) this plateau value is independent of the "history" of the adsorption process and corresponds well to the jamming limit predicted by the random sequential adsorption model; and (iv) surface denaturation, leading to enhanced surface binding and thus decreasing desorption constants, is the important phenomenon that can partly and qualitatively explain our observations. Its time dependence, however, remains to be clarified.

HREM study of irradiation damage in human dental enamel crystals.

Several phenomena have been observed during the examination of human dental enamel crystals (mainly constituted by hydroxyapatite (OHAP] by high-resolution electron microscopy (HREM) at 300 and 400 keV: orientation-dependent damage in the form of mass loss from voids or uniform destruction of crystal structure, beam-induced diffusion creating outgrowths at the crystal surfaces, recrystallization of the bulk crystal and crystallization of the inorganic components of the matrix surrounding the crystals. These beam-induced crystals have the CaO structure. The phenomena observed are most likely due to various electron-crystal interaction mechanisms (ballistic knock-on damage, electronic excitations, temperature rise, etc.). In this paper, the contribution of the ballistic process to the phenomena observed is discussed. The quantitative description of the knock-on collisions rests on the McKinley-Feshbach cross-section formula. The minimum ion displacement energies which appear in this expression have been estimated on the basis of the electrostatic ion binding energies, and the covalent bond energies if required. It is shown that hydroxyl, calcium and oxygen ions can effectively be displaced by the incident 300 and 400 keV electrons. Thus, the formation of CaO crystals by the combination of calcium and oxygen ions diffusing from their initial sites inside the OHAP lattice can tentatively be explained.

High-resolution electron microscopy of human enamel crystals.

The structure of enamel crystals obtained from four human premolars has been studied by high-resolution electron microscopy (HREM) in the [0001], [2110], [1540], [0110] and [1213] crystallographic directions at various microscope defocus and crystal thickness values. The resolution obtained has not previously been reported for human enamel crystals. In all cases, it was possible to match the experimental images to images calculated using the atomic positions of mineral hydroxyapatite. However, a deviation from hexagonal symmetry characterized by marked (1010) planes of intensity different from the one of the (3030) and (1010)-type planes was observed. In this work, we present an improvement of Scherzer resolution of 0.25-0.20 nm over previous work on biological enamel crystals. This improvement of resolution has permitted the incorporation of crystallographic reflections of higher spatial frequencies into the imaging process of the microscope and has led to a more precise structure determination of the crystals studied.

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.

Transmission electron microscopy of lattice planes in human alveolar bone apatite crystals.

Periodic fringes corresponding to six different lattice planes have been observed in apatite crystals of human normal alveolar bone by transmission electron microscopy. Three of these sets of fringes have spacings less than 3.5 A corresponding to the Scherzer resolution of the microscope used. The (0002) lattice plane of hydroxyapatite of 3.4 A d-spacings, the (2111) lattice plane with a d-spacing of 2.81 A, and the (3030) lattice plane with a d-spacing of 2.72 A have been identified. The (0002) and (2121) lattice planes have been observed for the first time in bone microcrystals. Some of the crystals studied were characterized by a mean width/thickness ratio of 6.91, typical of platelike habit, whereas observations of crystals aligned along the (1210) and (1211) directions showed a needlelike habit. The mean length of the bone apatite crystals was 470 A. A dark line similar to the one observed in enamel and dentine crystals was also seen. The bone microcrystals observed have shown a high sensitivity to beam damage.

Theoretical detection of a dark contrast line in twinned apatite bicrystals and its possible correlation with the chemical properties of human dentin and enamel crystals.

Electron microscope images of twinned apatite bicrystals oriented along the [1120] crystallographic direction have been simulated for various experimental conditions, and the validity of the calculation has been checked. These images show a dark contrast line similar to the one observed experimentally in enamel and dentin crystals and therefore strongly suggest the presence of a twin plane parallel to the (1100) crystallographic planes, in these crystals. The presence of a twin boundary in teeth and bone crystals is of prime importance for the adsorption and the dissolution properties of the calcified tissues as a whole.

High-resolution electron microscope and computed images of human tooth enamel crystals.

The structure of human enamel crystallites has been studied at a near atomic level by high-resolution electron microscopy. Electron micrographs have been obtained from crystallites present in human enamel with a structure resolution of 0.2 nm in the [0001], [1210], [1213], [1100] and [4510] zone axes directions. In most cases it was possible to match the experimental images with images calculated using the atomic positions of mineral hydroxyapatite. However, in some cases a discrepancy between calculated and experimental image detail was observed in the c direction of the [1210] and the [1100] images. This shows: (i) a structural heterogeneity of the crystals, and (ii) a loss of hexagonal symmetry of the structure. The resolution required to distinguish individual atomic sites in the different zones has been determined, and this will provide a useful basis for future work. As the determination of the "real structure" of biological crystals is of prime importance for the study of calcification mechanisms (crystal growth), biological properties and destructive phenomena of calcified tissues (i.e., dental caries and bone resorption).