Chemical & Nano-mechanical Study of Artificial Human Enamel Subsurface Lesions.

White lesions represent an early phase of caries formation. 20 human sound premolars were subjected to pH cycling procedure to induce subsurface lesions (SLs) in vitro. In addition, 2 teeth with naturally developed white spot lesions (WSLs) were used as references. All specimens characterized by confocal Raman microscopy being used for the first time in examining white & subsurface lesions and providing a high resolution chemical and morphological map based on phosphate peak intensity alterations at 960 cm-1. Nanoindentation technique was used to measure Hardness (H) and Young's modulus (E) of enamel. Phosphate map of examined samples exhibited presence of intact surface layer (ISL) followed by severe depletion in (PO43-) peak in the area corresponding to the body of the lesion. In all examined groups, the mechanical properties of enamel were decreased in lesion area and found to be inversely related to penetration depth of indenter owing to enamel hierarchical structure. By combining the above two techniques, we linked mechanical properties of enamel to its chemical composition and ensured that the two methods are highly sensitive to detect small changes in enamel composition. Further work is required to bring these two excellent tools to clinical application to perceive carious lesions at an early stage of development.

Laboratory Studies of Nonlinear Optical Signals for Caries Detection.

Multiphoton confocal microscopy and nonlinear spectroscopy are used to investigate the caries process in dentin. Although dentin is a major calcified tissue of the teeth, its organic phase comprises type I collagen fibers. Caries drive dentin demineralization and collagen denaturation. Multiphoton microscopy is a powerful imaging technique: the biological materials are transparent to infrared frequencies and can be excited to penetration depths inaccessible to 1-photon confocal microscopy. The laser excitation greatly reduces photodamage to the sole focal region, and the signal-to-noise ratio is improved significantly. The method has been used to follow pathologic processes involving collagen fibrosis or collagen destruction based on their 2-photon excited fluorescence (2PEF) emission and second harmonic generation (SHG). Combining multiphoton imaging with nonlinear spectroscopy, we demonstrate that both 2PEF and SHG intensity of human dentin are strongly modified during the tooth caries process, and we show that the ratio between SHG and 2PEF signals is a reliable parameter to follow dental caries. The ratio of the SHG/2PEF signals measured by nonlinear optical spectroscopy provides valuable information on the caries process, specifically on the degradation of the organic matrix of dentin. The goal is to bring these nonlinear optical signals to clinical application for caries diagnosis.

TEM study of the morphology of Mn2+ -doped calcium hydroxyapatite and beta-tricalcium phosphate.

Mn-doped carbonated hydroxyapatites (HA) were prepared by precipitation method. Ca-deficient HA samples were obtained by this method with the characteristic hexagonal apatite structure. Scanning transmission electron microscopy (STEM) of two HA samples with two different Mn content has shown that their morphology depends on their Mn content. In case of relatively low (0.73%) Mn content (HAMn1), platelet crystals about micron size and needle-like crystals up to 100 nm were observed, while with 1.23% Mn (HAMn2) crystals were smaller, needle-like and with sizes up to 400 nm only. Mn-doped TCP samples were prepared by two methods. In one case it was obtained by direct solid-state reaction with the characteristic rhombohedral structure of beta-TCP and with composition of Ca(2.7)Mn(0.3)(PO(4))(2). TEM pictures of crystals of this sample were tens of micron and submicron size with visible faces. Crystals of beta-TCP obtained by high temperature partial transformation of sample HAMn2 to beta-TCP were found by TEM to be smaller, micron sized, drop-like shaped, sensitive to beam radiation. These results indicate that the morphology of Mn doped beta-tricalcium phosphate samples depends on the method of their preparation. Morphological properties of HA and TCP are discussed and it is suggested that the smaller and less perfect HA crystals with the higher Mn-content as well as the less perfect TCP crystals obtained by transformation of HA to TCP might be of more biocompatible character.

Immediate implant placement using injectable calcium phosphate hydraulic cement in dogs.

Techniques allowing implant placement in extraction sockets require either high diameter implants in surgically enlarged sockets or grafting and/or regenerative procedures around implants after their primary surgical stabilization. This study aimed to evaluate the ability of calcium phosphate hydraulic cement (CPHC) to immobilize commercially available titanium implants in extraction sockets. CPHC was used in seven fresh dog extraction sockets in conjunction with ITI TPS implants. Three extraction sockets without CPHC were used as controls. Initial implant stability was measured after 10 min hardening with periostest. The dogs were sacrificed after 9 months. Non-decalcified specimens were prepared for histologic and histomor-phometric examination. The surface percentage of implant-to-mineralized bone contact and bone density was calculated for each specimen. The periotest values were significantly different for implants stabilized with CPHC than for the controls, and simi-lar to values reported for osteointegrated implants. New alveolar bone was formed in intimate contact with titanium. In two cases, non-resorbed CPHC residues were observed closely bound to the implant. This study clearly demonstrates that CPHC cement is suitable for immediate implant immobilization in extraction sockets.

Injectable calcium phosphate hydraulic cement (CPHC) used for periodontal tissue regeneration: A study of a dog model.

Injectable calcium phosphate hydraulic cement (CPHC) is a new bone substitute family. This study aimed to evaluate the use of CPHC in surgical periodontitis-simulating defects in a dog model. CPHC was obtained by adding powder mixtures of different calcium phosphates with different solubility. Alveolar bone was removed by drilling over the mesial and distal roots of the 2nd mandibular premolar in six dogs. The defects were randomly selected, three were untreated and six treated. The defects had a depth of 6 mm and a width of 3 mm. The animals were sacrificed after 9 months and samples prepared, with no decalcification, for histological evaluation. Seventy-nine percent of the root was covered by bone in the experimental defects, compared to 41% of the root for the control defects. Bone height was significantly higher for the experimental defects (4.9 +/- 0.9 mm) than for the control defects (1.4 +/- 0.5 mm). After 9 months, 97 +/- 6% of the CPHC was degradated and replaced by bone. This study proves the interest of this cement because of the particularly high level of periodontal bone regeneration. The ability of the cement to be easily injected and shaped in bone defects and the immediate immobilization of the teeth after hardening is notable. (Journal of Applied Biomaterials & Biomechanics 2003; 1: 186-93).

High-resolution electron-microscopic study of the relationship between human enamel and dentin crystals at the dentinoenamel junction.

The development of dentin and of enamel share a common starting locus: the dentinoenamel junction (DEJ). In this study the relationship between enamel and dentin crystals has been investigated in order to highlight the guiding or modulating role of the previously mineralized dentin layer during enamel formation. Observations were made with a high-resolution electron microscope and, after digitalization, image-analysis software was used to obtain digital diffractograms of individual crystals. In general no direct epitaxial growth of enamel crystals onto dentin crystals could be demonstrated. The absence of direct contact between the two kinds of crystals and the presence of amorphous areas within enamel particles at the junction with dentin crystals were always noted. Only in a few cases was the relationship between enamel and dentin crystals observed, which suggested a preorganization of the enamel matrix influenced by the dentin surface structure. This could be explained either by the existence of a proteinaceous continuum between enamel and dentin or by the orientation of enamel proteins by dentin crystals.

Carbonated hydroxyapatites precipitated in the presence of Ti.

Hydroxyapatites (HA) were prepared by precipitation from an aqueous solution with Ti4+ (0-2500 microg/g) and with carbonate (0.8-4.0%) at pH 7.0. The uptake of Ti was found to be 75% of the original amounts. Stoichiometric ratios of Ca/P (1.67) were found for low carbonate samples. X-ray diffraction and IR spectroscopy have shown that samples have structural data characteristic for HA. Heat treatment and thermogravimetric analysis (20-900 degrees C) have shown carbonate decomposition enhanced by the presence of Ti and no transformation of the HA structure. It was also found that 0.2 mol of adsorbed and 0.6-0.8 mol of crystalline water are released from the samples during heating. Transmission electron microscopy revealed the formation of plate like crystals which increase in size with increase of carbonate content. Samples with high carbonate and high Ti content have irregular shape and are sensitive to electron beam irradiation as opposed to non-doped samples. Ti appears to have a destabilizing effect on HA. The incorporation of Ti in HA and the biological relevance of Ti in bone crystals is discussed.

High-resolution electron microscopy study of synthetic carbonate and aluminum containing apatites.

Aluminum (Al)-containing calcium-deficient carbonated hydroxyapatites were produced by a precipitation method from aqueous solution with carbonate (0-6.1%) and aluminum (0.1-0.5%) concentrations close to those found in biological materials. Two series of apatites were prepared: one at pH 7.0 and another at pH 9. 0. High-resolution electron microscopy has shown that many of them possess structural defects such as screw dislocations, grain boundaries, and central defects. Samples with high carbonate content and high water and high Al(3+) content had a high amount of structural defects. Accordingly, a sample (7Al1) with a relatively high carbonate content (6.1%) and a sample (7Al6) without carbonate but with a relatively high water (2.0 mol) and Al(3+) content (0. 39%) presented the highest amount of structural defects, 54% and 47%, respectively. A sample (7Al13) with a low level of crystalline water (1 mol) and low carbonate (2.5%) showed a small amount of defects. The presence of water associated with Al(3+) induced a high number of crystals having a central defect with a great similarity to the so-called water layer of octacalcium phosphate (OCP). Observed images of all these crystals have shown good correspondence with the computer-simulated image based on the crystal structure of hydroxyapatite, indicating that the addition of Al(3+) and carbonate does not perturb the apatitic structure.

Protein interactions with polyelectrolyte multilayers: interactions between human serum albumin and polystyrene sulfonate/polyallylamine multilayers.

The interactions between polystyrenesulfonate (PSS)/polyallylamine (PAH) multilayers with human serum albumin (HSA) were investigated by means of scanning angle reflectometry (SAR). We find that albumin adsorbs both on multilayers terminating with PSS (negatively charged) or PAH (positively charged) polyelectrolytes. On films terminating with PSS only, an albumin equivalent monolayer is found whereas when PAH constitutes the outer layer, albumin interacts with the multilayer in such a way as to form a protein film that extends over thicknesses that can be as high as four times the largest dimension of the native albumin molecule. Once the protein film is formed, it is found that when the albumin solution is replaced by a pure buffer solution of same ionic strength as the adsorption solution almost no desorption takes place. On the other hand, when a buffer solution of higher ionic strength is brought in contact with the albumin film, a significant amount of adsorbed proteins is released. One also observes that, for albumin solutions of a given protein concentration, the adsorbed protein amount depends on the ionic strength of the adsorption solution. On surfaces terminating with PAH, the adsorbed protein amount first increases rapidly but passes through a maximum and decreases with the ionic strength. The ionic strength corresponding to the maximum of the adsorbed albumin amount itself depends on the albumin concentration. On the other hand, on films terminating with PSS the adsorbed amount increases with the salt concentration before leveling-off. These results show that the underlying complexity of concentration and pH dependent adsorption/desorption equilibria often simply termed "protein adsorption" is the result of antagonist competing interactions that are mainly of electrostatic origin. We also propose two microscopic models, that are compatible with our experimental observations.

Initial aspects of mineralization at the dentino-enamel junction in embryonic mouse incisor in vivo and in vitro: a tem comparative study.

The frontier between the enamel organ and the dental papilla, the future dentino-enamel junction, undergoes coordinated modifications. The mineralization of the extracellular matrix starts within the predentine, which is a prerequisite for the formation of the first enamel crystallites in vivo. We investigated the dentino-enamel junction using the embryonic mouse incisor as a model. Our data showed that the notion of the dentino-enamel junction should not be restricted to the thin interface classically described. A temporo-spatial survey from the epithelio-mesenchymal junction to the dentino-enamel junction delineated a clear sequence of events characterized by the early deposition of electron-dense granules, followed by the appearance of patches of stippled material at the dentino-enamel junction. The first tiny enamel crystallites appeared in the vicinity of this material which presented a well-ordered alignment. The comparison of data obtained in vivo on 17-, 18-, 19-d-old embryonic incisors with those obtained in vitro using 15-d-old embryonic incisors cultured for 7 d emphasizes the relevance of this sequence. Helicoidal growing crystals were observed in cultured tooth germs but never in vivo.

First experimental evidence for human dentine crystal formation involving conversion of octacalcium phosphate to hydroxyapatite.

Biological apatite-crystal formation is a complex process starting with heterogeneous nucleation of inorganic calcium phosphate on an organic extracellular matrix [Cuisinier et al. (1995), J. Cryst. Growth, 156, 443-453]. Further stages of crystal growth are also controlled by the organic matrix and both nucleation and growth processes are under cellular control [Mann (1993), Nature (London), 367, 499-505]. The final mineral in calcified tissue is constituted by poorly crystalline hydroxyapatite (HA) with a low Ca:P ratio, containing foreign ions such as carbonate and fluoride. This study reports the first observation of octacalcium phosphate (OCP) [Brown (1962), Nature (London), 196, 1048-1055] in a biological tissue; OCP was found in the central part and HA at the extremities of the same crystal of calcifying dentine. This observation is of key importance in understanding the first nucleation steps of biological mineralization. The presence of OCP in a forming human dentine crystal and the observation in the same tissue of nanometer-sized particles with a HA structure [Houllé et al. (1997), J. Dent. Res. 76, 895-904] clearly proves that two mechanisms, direct nucleation of non-stoichiometric HA crystals and nucleation of OCP, occur simultaneously in same area of mineralization. OCP is found to be a transient phase during the growth of biological crystals. In small crystals, OCP is completely transformed into HA by a hydrolysis reaction (Brown, 1962) and can only be detected in larger crystals because of its slow kinetics of transformation.

High resolution electron microscopy: structure and growth mechanisms of human dentin crystals.

Biological crystal formation was postulated to begin by a nucleation process. Such processes have been demonstrated for human amelogenesis and bone mineralization. The aim of this study was to confirm if such mechanisms occur during dentin crystal formation. The structure of human fetal dentin crystals and the earliest stages of mineral growth were followed by High Resolution Electron Microscopy (HREM) associated with digitalized image analysis. Micrographs of the mineralization front were first digitalized, and selected areas were transformed in the reciprocal space by Fast Fourier Transform. The resulting diffractograms were compared with computer-simulated diffractograms and used to determine the orientation of crystals. Dentin crystals, found close to the mineralization front, show a structure closely related to that of hydroxyapatite (HA), as determined by comparison of HREM images with simulated images. These crystals present numerous structural defects such as dislocations and grain boundaries. These defects appear to be present in dentin crystals at an early stage of growth. We have also observed nanometer-sized particles in mineralization areas. Calculated diffractograms of these areas show significant similarities with HA diffraction patterns, and in one case, their structure could be correlated to HA structure through an image simulation process. These nanometer-sized particles could be related to the nucleation process, and their growth, orientation, and formation appear to be mediated by extracellular matrix components.

HRTEM study of biological crystal growth mechanisms in the vicinity of implanted synthetic hydroxyapatite crystals.

Calcium phosphates are widely used as biomaterials. Ultrastructural assessments are of the utmost importance in our understanding of interfacial phenomena. The aim of this study was to learn more about the newly formed crystal growth mechanisms. The interfaces between implanted synthetic hydroxyapatite crystals (HAS) and newly formed crystallites were thoroughly examined on a molecular level. The bone-grafting material (HAS) was implanted into two adult patients, and small biopsies were recovered 6 months after implantation. The raw biomaterial was analyzed by x-ray diffraction and high-resolution transmission electron microscopy (HRTEM). Six months after their implantation, the HAS aggregates were surrounded by a mineralized bone matrix. Tiny crystallites also filled the spaces between the HAS crystals within the aggregates. These newly formed crystallites growing at the surfaces of the implanted HAS crystals appeared to be apatitic. The crystallographic investigations of the nucleation and growing mechanisms of the newly formed crystallites were performed by HRTEM in association with computer simulation and mathematical processing of digitized images. A relationship was noted between the orientation axes of crystallites growing nearby and the zone axes of the implanted HAS, thus strongly suggesting a guiding or substratum role of the HAS particles.

Adhesion of hard spheres under the influence of double-layer, van der Waals, and gravitational potentials at a solid/liquid interface.

The deposition process of colloidal particles or microorganisms on flat surfaces is analyzed by means of computer simulations. Interparticle interactions (double layer and van der Waals) and weak gravitational forces are taken into account; hydrodynamic interactions, on the other hand, are neglected. In particular, the deposition probability as a function of the deposition location of a particle in the presence of one or two identical fixed particles is discussed. It is shown, in particular, that the ratio of the adhesion probabilities at a given location r, for particles subject to weak gravitation, in the presence and in the absence of the interparticle interaction U(r) follows approximately a Boltzmann law exp[-U(r)/kT], even though the adsorption process is fully irreversible. This result validates, as far as the distribution function of particles on a surface is concerned, Adamczyk's assumption [Adamczyk, Z., Zembala, M., Siwek, B. & Warszynski, P. (1990) J. Colloid Interface Sci. 140, 123-137] that the adhesion process of Brownian particles can be modeled by a random sequential adsorption model with an adsorption probability equal to exp[-U(r)/kT].

Atomic force microscopy study of human tooth enamel surfaces.

Human enamel features from individual crystals up to prisms were observed by atomic force microscopy (AFM). Low magnification images of vestibular tooth surfaces show the existence of enamel prisms appearing as deep holes. Individual, parallel enamel crystals show lateral faces elongated and formed by the (100) planes of hydroxyapatite (HA). Height differences between (001) faces create the roughness of enamel surface. Individual (001) crystal faces can be observed clearly at higher magnification and show the characteristic hexagonal shape with 60 degree angles between (100) faces. This study confirms the applicability of AFM for studying biological hydroxyapatite crystals.

Influence of diffusion and gravity on the adhesion of a two-component mixture of hard spheres on a flat surface.

The adhesion of hard spheres, modeling particles of biological interest (proteins, bacteria, cells), on flat surfaces is investigated by means of Monte Carlo simulations. These computations include the Brownian diffusion of the particles in the bulk fluid, as well as the systematic displacement due to the gravitational field. The size of the particles influences directly both diffusion coefficient and net weight, with the consequence that the coverage at the jamming limit depends on this parameter. Results obtained in a former paper based on a lattice model are confirmed by the present continuous space model. In order to gain a better understanding of the adsorption competition of two types of particles, the proposed model is applied to the case of binary mixtures of spheres. For polydispersed suspensions, various parameters determine the final coverage, as well as the distribution of the small and large particles on the surface: the radii of the particles and the respective proportions of them in the infinitely large reservoir from which they are randomly selected. In this way, it is shown that the chronology of the adhesion of the small and large particles strongly influences the final number of each type of spheres fixed on the surface. Qualitatively, the present results resemble those obtained with disks placed by means of a classical random sequential adsorption mechanisms. Quantitatively, however, the number densities and coverage values determined in this way are significantly different due to the inclusion of the gravity and of the diffusion in the model.

Secnidazole concentrations in plasma and crevicular fluid after a single oral dose.

Metronidazole and related nitroimidazole derivatives, including ornidazole and tinidazole, have been used successfully in the treatment of periodontal diseases. The purpose of this study was to measure secnidazole (another nitroimidazole derivative) concentrations in plasma and gingival crevicular fluid (GCF) after intake of a single oral dose. Secnidazole concentrations were estimated in 11 human healthy volunteers after a single dose of 2 g taken orally. Samples of blood and GCF were collected before intake and during the following 72 h. A high-performance liquid chromatography (HPLC) method has been developed for the determination of secnidazole in microsamples (1 to 3 microliters) of GCF. The mean peak blood and GCF levels were equal to 40.5 +/- 9.4 micrograms ml-1 at 2 h in blood and 26.4 +/- 7.0 micrograms ml-1 at 1 h in GCF, respectively. Apparent elimination half-life was 28.8 h (blood) and 30.4 h (GCF), respectively. These results show that the rate and extent-absorption of secnidazole are slightly higher in blood than in GCF, but the elimination of the drug is similar in the two body fluids.

Human amelogenesis: high resolution electron microscopy of nanometer-sized particles.

Growth of inorganic crystals of enamel is described as a two-stage process with growth of ribbon-like crystals in length and width, followed by their development in thickness. In early stages of crystal growth during human amelogenesis nanometer-sized particles with a mean diameter of 1.1 nm were described between ribbon-like crystals. These small particles had a crystalline structure but their lattice parameters did not seem to be directly related to those of calcium phosphates. The nanometer-sized particles appear to correspond to initial stages of apatite crystal growth. Their localization close to ribbon-like crystals and their progressive increase in size and number may indicate that they represent a precursor phase for these crystals. Nucleation areas at both extremities, of elongated ribbon-like crystals could be involved in the two-directional growth of ribbons and/or in nanometer-sized particle nucleation.

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.

Human amelogenesis. I: High resolution electron microscopy study of ribbon-like crystals.

Ribbon-like crystals, from developing enamel of human fetuses, were studied by high resolution electron microscopy. These crystals were classically described as the first organized mineral formed during amelogenesis. They were characterized by a mean width-to-thickness ratio (W.T-1) of 9.5, and 40% were bent. On lattice images we noted the presence of the central dark line (CDL) associated with white spots. Both structures were found in crystals with a minimum thickness of 8-10 nm. CDLs were localized in the center of the crystals and seemed to be linked to the initial growth process, but their exact structure and function were not fully determined. We were able to study the structure of the ribbon-like crystals with a Scherzer resolution close to 0.2 nm. The good correspondence between experimental and computed images showed that their structure was related to hydroxyapatite (HA). In addition, the presence of ionic substitutions and deficiencies were also compatible with HA. In this study, about 50% of the crystals showed structural defects. Screw dislocations were the most often noted defects and were observed within crystals aligned along five different zone axes. Low- and high-angle boundaries were also detected. Low-angle boundaries, found in the center of the crystals, could thus be related to CDLs and be implicated in the nucleation step of crystal formation, whereas high-angle boundaries could result from the fusion of ribbon-like crystals. Such mechanisms could induce an acceleration of the growth in thickness of the crystal observed during the maturation stage of amelogenesis.