Characterization of rat vertebrae cortical bone microstructures using confocal Raman microscopy combined to tomography and electron microscopy.

BACKGROUND: The rat vertebrae is a good model to study bone regeneration after implantation of biomaterials used to treat bone loss, a major problem in oral and dental surgery. However, the precise characterization of bone microstructures in the rat vertebrae has not been reported. Therefore, the aim of this study was to achieve the complete analysis of such bone, at different scales, in order to have a clear model of healthy bone for comparison with regenerated bone. METHODS: In order to image the cortical bone of rat caudal vertebra, confocal Raman microscopy was combined with high resolution X-ray micro computed tomography (micro-CT), with scanning electron microscopy (SEM) using backscatter electron imaging and with more conventional histology coloration techniques. SEM and Raman microscopy were done in various regions of the cortical bone corresponding to external, middle and internal areas. The spongy bone was imaged in parallel. Micro-CT was performed on the whole vertebra to monitor the network of haversian canals in the cortical bone. Osteonic canals characteristics, and relative chemical composition were analysed in several regions of interest, in cortical and spongy bone. Five rats were included in this study. RESULTS: On micro-CT images, differences in intensity were observed in the cortical bone, substantiated by SEM. Chemical analysis with Raman spectra confirmed the difference in composition between the different regions of the cortical and spongy bone. PCA and k-mean cluster analysis separated these groups, except for the external and middle cortical bone. Peak intensity ratio confirmed these results with a CO3 to ν2 PO4 ratio significantly different for the internal cortical bone. Grayscale images stack extracted from micro-CT showed that global architecture of cortical bone was characterized by a dense and complex network of haversian osteonic canals, starting from the surface towards the vertebrae center. The mean diameter of the canals was 18.4 µm (SD 8.6 µm) and the mean length was 450 µm (SD 152 µm). Finally, Raman reconstructed images of the lamellar bone showed an enlargement of the lamellar layer width, both in circumferential lamellar bone and around haversian canals. CONCLUSIONS: Micro-CT and confocal Raman microscopy are good tools to complete classical analysis using optical and electron microscopy. The results and measurements presented in a rat model known for its small inter-individual differences provide the main characteristics of a mature bone. This study will allow the community working on this rat vertebrate model to have a set of characteristics, in particular on the structure of the haversian canals.

Human tooth enamel tuft drapes revealed by microtomography.

OBJECTIVE: The tufts of human dental enamel are structures located at the enamel-dentin junction and whose origin has not been clearly established. Although studies have highlighted their protein content and hypomineralization, none has been able to shed light on their 3D structure. The aim of this study was to reveal the whole structure using high-resolution conventional microtomography. DESIGN: Ten adult mandibular first and second molars and two primary mandibular first molars were sectioned and scanned with microcomputed tomography with a resolution between 4.7 and 5 micrometers. By determining the threshold discriminating dentin and tufts, we were able to reconstruct 3D meshes. RESULTS: We revealed the exact pattern of the tufts in adult molars and discovered their distribution, their dynamics, and the existence of a regular undulation, forming a particular angle of approximately 30 degrees with the dentin surface. A spatial frequency of approximately 160 micrometers would be compatible with the variation in the orientation of groups of dental enamel rods. In contrast, the present setting is not sufficient to extract similar information for primary teeth. CONCLUSIONS: Enamel tufts have a specific pattern, with an oriented draped form and are regularly spaced. The possible connection between these undulations and the Hunter-Schreger bands (diazonias and parazonias) needs to be studied.

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.

A preliminary study of a new endodontic irrigation system: Clean Jet Endo.

The purpose of the present study was to assess the irrigant penetration and cleaning ability of a new irrigation system, the Clean Jet Endo (Produits Dentaires SA, Switzerland) in comparison to conventional irrigation followed or not by sonic activation. Irrigant penetration was evaluated on resin blocks simulators by measuring the methylene blue absorbance thanks to a UV/visible spectrophotometer and cleaning ability was assessed in an ex vivo experiment according to the debris score in an artificial canal extension before and after the final irrigation protocol. A statistical analysis was carried out in order to highlight the significant differences between the irrigation techniques. Clean Jet Endo permitted to better eliminate the methylene blue into the simulated canals. A significant difference between the 2 techniques was observed in the middle third (p = 0.005) as well as in the apical third (p < 0.2). An additional microscope observation (16X) confirmed that Clean Jet Endo@ usage led to a better penetration of irrigant within the lateral canals of the simulators. Likewise, this irrigating system permitted to better eliminate the debris in the lateral groove than the other techniques. In conclusion, our findings implied the potential of this new irrigation system to enhance root canal debridement and disinfection.

Use of new minimum intervention dentistry technologies in caries management.

Preservation of natural tooth structure requires early detection of the carious lesion and is associated with comprehensive patient dental care. Processes aiming to detect carious lesions in the initial stage with optimum efficiency employ a variety of technologies such as magnifying loupes, transillumination, light and laser fluorescence (QLF® and DIAGNOdent® ) and autofluorescence (Soprolife® and VistaCam®), electric current/impedance (CarieScan(®) ), tomographic imaging and image processing. Most fluorescent caries detection tools can discriminate between healthy and carious dental tissue, demonstrating different levels of sensitivity and specificity. Based on the fluorescence principle, an LED camera (Soprolife® ) was developed (Sopro-Acteon, La Ciotat, France) which combined magnification, fluorescence, picture acquisition and an innovative therapeutic concept called light-induced fluorescence evaluator for diagnosis and treatment (LIFEDT). This article is rounded off by a Soprolife® illustration about minimally or even non-invasive dental techniques, distinguishing those that preserve or reinforce the enamel and enamel-dentine structures without any preparation (MIT1- minimally invasive therapy 1) from those that require minimum preparation of the dental tissues (MIT2 - minimally invasive therapy 2) using several clinical cases as examples. MIT1 encompasses all the dental techniques aimed at disinfection, remineralizing, reversing and sealing the caries process and MIT2 involves a series of specific tools, including microburs, air abrasion devices, sonic and ultrasonic inserts and photo-activated disinfection to achieve minimal preparation of the tooth. With respect to minimally invasive treatment and prevention, the use of lasers is discussed. Furthermore, while most practices operate under a surgical model, Caries Management by Risk Assessment (CaMBRA) encourages a medical model of disease prevention and management to control the manifestation of the disease, or keep the oral environment in a state of balance between pathological and preventive factors. Early detection and diagnosis and prediction of lesion activity are of great interest and may change traditional operative procedures substantially. Fluorescence tools with high levels of magnification and observational capacity should guide clinicians towards a more preventive and minimally invasive treatment strategy.

Factors affecting the viscosity of sodium hypochlorite and their effect on irrigant flow.

AIM: To assess the influence of concentration, temperature and surfactant addition to a sodium hypochlorite solution on its dynamic viscosity and to calculate the corresponding Reynolds number to determine the corresponding flow regimen. METHODOLOGY: The dynamic viscosity of the irrigant was assessed using a rotational viscometer. Sodium hypochlorite with concentrations ranging from 0.6% to 9.6% was tested at 37 and 22 °C. A wide range of concentrations of three different surfactants was mixed in 2.4% sodium hypochlorite for viscosity measurements. The Reynolds number was calculated under each condition. Data were analysed using two-way anova. RESULTS: There was a significant influence of sodium hypochlorite concentration (P < 0.001) and temperature (P < 0.001) on dynamic viscosity: the latter significantly increased with sodium hypochlorite concentration and decreased with temperature. A significant influence of surfactant concentration on dynamic viscosity (P < 0.001) occurred, especially for high surfactant concentrations: 6.25% for benzalkonium chloride, 15% for Tween 80 and 6.25% for Triton X-100. Reynolds number values calculated for a given flow rate (0.14 mL s(-1)), and root canal diameter (sizes 45 and 70) clearly qualified the irrigant flow regimen as laminar. CONCLUSIONS: Dynamic viscosity increased with sodium hypochlorite and surfactant concentration but decreased with temperature. Under clinical conditions, all viscosities measured led to laminar flow. The transition between laminar and turbulent flow may be reached by modifying different parameters at the same time: increasing flow rate and temperature whilst decreasing irrigant viscosity by adding surfactants with a high value of critical micellar concentration.

Human dental pulp stem cells growth and osteodifferentiation on porous resorbable scaffolds.

Short Communication selected from the Oral Presentations of the 56th Congress of the Groupèment International pour la Recherche Scientifique en Stomatologie et Odontologie, Peñafiel (Portugal) May 2012.

[Not Available]. / Fonctionnalisation du Titane par des Peptides Specifiques Bi-Fonctionnels pour l'adhesion Epitheliale.

Short Communication selected from the Oral Presentations of the 56th Congress of the Groupèment International pour la Recherche Scientifique en Stomatologie et Odontologie, Peñafiel (Portugal) May 2012.

Biomimetic organic-inorganic nanocomposite coatings for titanium implants. In vitro and in vivo biological testing.

Recently described organic-inorganic nanocomposite coatings of the chemical composition: (PLL/PGA)(10)CaP[(PLL/PGA)(5)CaP](4) (coating A) and (PLL/PGA)(10)CaP[(PLL/PGA)(5)CaP](4)(PLL/PGA)(5) (coating B), applied to chemically etched titanium plates, have been tested by extensive cell culture tests and in vivo biological experiments, with uncoated titanium plates serving as controls. Before testing, coated samples were stored for extended periods of time (from 2 weeks to 8 months) under dry, sterile conditions. Cells of the cell-lines MC3T3-E1 and/or SAOS-2 were used for the following cell culture tests: initial adhesion (4 h) and proliferation (up to 21 days), cell activity (XTT test), morphology, synthesis of collagen type I and alkaline phosphatase activity (all incubation up to 21 days). In addition, coating B was tested against uncoated control in a validated in vivo pull-out model in rabbit tibia. The results of both in vitro and in vivo experiments show excellent biological properties of chemically etched titanium which are even surpassed by surfaces covered with coating B. Thus, after 8 weeks of healing the implants coated with B were significantly better attached to the cortical bone of rabbit thibiae than uncoated titanium controls with more than twice the force needed to detach coated implants. However, coating A (top crystal layer) had an adverse effect on both cell proliferation and activity, which is explained by morphological observations, showing inhibited spreading of the cells on its rough surfaces. The results also show the remarkable stability of the coatings when shelved under dry and sterile conditions.

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.

Human osteoblast response to pulsed laser deposited calcium phosphate coatings.

Octacalcium phosphate (OCP) and Mn(2+)-doped carbonate hydroxyapatite (Mn-CHA) thin films were deposited on pure, highly polished and chemically etched Ti substrates with pulsed laser deposition. The coatings exhibit different composition, crystallinity and morphology that might affect their osteoconductivity. Human osteoblasts were cultured on the surfaces of OCP and Mn-CHA thin films, and the cell attachment, proliferation and differentiation were evaluated up to 21 days. The cells showed a normal morphology and a very good rate of proliferation and viability in every experimental time. Alkaline phosphatase activity was always higher than the control and Ti groups. From days 7 to 21 collagen type I production was higher in comparison with control and Ti groups. The level of transforming growth factor beta 1 (TGF-beta1) was lower at 3 and 7 days, but reached the highest values during following experimental times (14 and 21 days). Our data demonstrate that both calcium phosphate coatings favour osteoblasts proliferation, activation of their metabolism and differentiation.

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.