Nano-atomic scale hydrophobic/philic confinement of peptides on mineral surfaces by cross-correlated SPM and quantum mechanical DFT analysis.

The fundamental knowledge of the interaction between biomolecules and mineral surfaces is of utmost importance to drive new technological advancements, particularly for condensation, aggregation, catalysis and exchange of biomolecules. The mineral surface can be used in several fields and applications, for instance in biotechnology, environmental science and remediation, soil science, agro-food and related technology. This kind of knowledge may also provide several suggestions and have implications also for the prebiotic chemistry field, namely the study of the abiotic physicochemical steps that could have led to the 'creation' of the first known living organism. Nowadays, this kind of information at the micro and nanometric scale can be explored with several experimental and theoretical techniques and, among them, atomic force microscopy (AFM)-related methods and density functional theory (DFT) are particularly suited to investigate adsorption processes at single molecule level. In the present work, the specific interaction at the atomic scale between a small peptide (di-glycine) and the (001) surface of clinochlore, a mineral presenting alternately stacked talc-like layers (hydrophilic) and brucite-like sheets (hydrophobic), was characterized by means of a cross-correlated approach combining AFM and DFT simulations. The experiments evidenced the preferential adsorption of di-glycine onto the hydrophobic brucite-like sheet of the mineral, with the observed molecules organized as dot-like (single-molecules), agglomerates, filament-like and network structures by the surface, whereas only very few peptides were imaged onto the hydrophilic talc-like layer. From the theoretical analysis, the most stable conformation of the di-glycine peptide adsorbed on the mineral surface was calculated, and the binding energy analysis of the specific interaction of the molecule, depending on the local chemistry of the substrate, provided fundamental information to interpret end explain the experimental evidence. LAY DESCRIPTION: The present work aims at extending the knowledge of the biomolecules/minerals interaction world. The fundamental knowledge of the interaction between biomolecules and mineral surfaces is of utmost importance to drive the development of new technological advancements, particularly for condensation, aggregation, catalysis and exchange of biomolecules. The mineral surface can be used as substrate in several fields and applications, for instance in biotechnology, environmental science and remediation, soil science, agro-food and related technology. This kind of research may also provide several suggestions and have implications also for the prebiotic chemistry field, namely the study of the abiotic physicochemical steps that could have led to the "creation" of the first known living organism. Nowadays, this kind of research at the micro and nanometric scale can be performed with several experimental techniques and, among them, scanning probe microscopy-related methods are particularly suited to investigate surface adsorption processes at single molecule level. In the present work, the focus is on the specific interaction at the atomic scale between a small peptide (di-glycine) and the surface of clinochlore (a diffuse clay mineral). Clinochlore is a mineral belonging to the phyllosilicate, formed by alternately stacked hydrophilic talc-like layers [chemical formula Mg3 Si4 O10 (OH)2 ] and hydrophobic brucite-like sheets [magnesium hydroxide, Mg(OH)2 ]. Since these two kind of layered structures are held together by weak (van der Waals) forces, the mineral can be easily cleaved along the [001] crystallographic direction (the stacking direction) and the resulting (001) clinochlore surface may presents at the nanoscale some remainders of one layer (brucite-like or talc-like) on the other. This means that this mineral exposes to the environment two different type of surfaces, one hydrophilic and one hydrophobic, which can selectively interact with (adsorb) different type of molecules at the nanoscale. Clinochlore is also one of the 420 mineral species that were likely present on Earth at the time of life's origins, thus it could have played a fundamental role in prebiotic chemistry. In this study, the interaction between di-glycine and clinochlore was characterized by means of both atomic force microscopy (AFM) at the nanometric scale and density functional theory (DFT) simulations, correlating the results of the two methods (cross-correlation approach). The experiments evidenced the preferential adsorption of di-glycine onto the hydrophobic brucite-like sheet of the mineral, with the observed molecules organized as dot-like (single-molecules) structures, agglomerates, filaments and networks by the surface, whereas only very few peptides were imaged onto the hydrophilic talc-like layer. From the theoretical analysis, the most stable conformation of the di-glycine adsorbed on the mineral surface was calculated, and the binding energy analysis of the specific interaction of the molecule, depending on the local chemistry of the substrate, provided fundamental information to interpret end explain the experimental evidence.

Effects of stannous fluoride on eroded enamel permeability.

This study aimed to evaluate the effect in vitro of a single application of a stannous fluoride- (SnF2-) containing toothpaste on eroded enamel. Forty-eight teeth were subjected to three acid treatments: 15% hydrochloric acid for 120 s (HA group); 1% citric acid (pH=4) for 180 s (CA group); 37% phosphoric acid for 30 s (PA group). They were brushed with an electric toothbrush with pressure control and 1 g of SnF2 (1100 ppm) toothpaste for 2 min. Polyether replicas of buccal enamel surfaces were obtained at baseline, after acid exposure and after brushing, gold sputtered and inspected by SEM for fluid droplets presence. Hydrochloric and citric acid treatments increased enamel permeability while, on the contrary, phosphoric acid reduced enamel fluid release. SnF2 application of ameliorated acid induced permeability in citric and hydrochloric treated samples. Permeability in phosphoric treated enamel was unchanged after topical application of SnF2. Our data show specific acid-dependent effects on enamel permeability and demonstrate that SnF2 application can reverse acid-induced permeability.

In vitro effects of fluoride-based and desensitizing toothpastes on dentine permeability.

This study aims to evaluate the effectiveness in reducing dentine permeability of three different toothpastes comparing with a sodium fluoride one. Dentine permeability was measured by a fluid filtration system. Dentin discs were randomized in four experimental groups and treated with different commercial toothpastes, as it follows. Group A: Sensodyne® Rapid, with strontium acetate; group B: Elmex Sensitive ProfessionalTM, with arginine and 450 ppm monofluoro-phosphate; group C: Colgate Total Advanced®, with 1450 ppm NaF; group D: AZ Pro-Expert®, with 1100 ppm SnF2. After brushing specimens of each groups were assigned to two subgroups and: 1) stored in artificial saliva (2 h) and treated with 6% citric acid for 1 min; 2) stored in artificial saliva for 24 h. SEM analysis was performed to investigate dentinal tubules occlusion of acid treated and stored in artificial saliva samples. Dentin permeability proved affected by dentifrice treatments. The toothpastes specifically formulated for hypersensitivity showed significant effects concerning decrease of dentine permeability. SEM observations demonstrated the presence of dentifrice particles on dentin surface and inside dentin tubules. Stannous fluoride treated samples exhibited the greater tubules occlusion.

Experimental etch-and-rinse adhesives doped with bioactive calcium silicate-based micro-fillers to generate therapeutic resin-dentin interfaces.

OBJECTIVES: This study aimed at evaluating the therapeutic bioactive effects on the bond strength of three experimental bonding agents containing modified Portland cement-based micro-fillers applied to acid-etched dentin and submitted to aging in simulated body fluid solution (SBS). Confocal laser (CLSM) and scanning electron microscopy (SEM) were also performed. METHODS: A type-I ordinary Portland cement was tailored using different compounds such as sodium-calcium-aluminum-magnesium silicate hydroxide (HOPC), aluminum-magnesium-carbonate hydroxide hydrates (HCPMM) and titanium oxide (HPCTO) to create three bioactive micro-fillers. A resin blend mainly constituted by Bis-GMA, PMDM and HEMA was used as control (RES-Ctr) or mixed with each micro-filler to create three experimental bonding agents: (i) Res-HOPC, (ii) Res-HCPMM and (iii) Res-HPCTO. The bonding agents were applied onto 37% H3PO4-etched dentin and light-cured for 30s. After build-ups, they were prepared for micro-tensile bond strength (µTBS) and tested after 24h or 6 months of SBS storage. SEM analysis was performed after de-bonding, while CLSM was used to evaluate the ultra-morphology/nanoleakage and the mineral deposition at the resin-dentin interface. RESULTS: High µTBS values were achieved in all groups after 24h. Only Res-HOPC and Res-HCPMM showed stable µTBS after SBS storage (6 months). All the resin-dentin interfaces created using the bonding agents containing the bioactive micro-fillers tested in this study showed an evident reduction of nanoleakage and mineral deposition after SBS storage. CONCLUSION: Resin bonding systems containing specifically tailored Portland cement micro-fillers may promote a therapeutic mineral deposition within the hybrid layer and increase the durability of the resin-dentin bond.

SPM nanolithography of hydroxy-silicates.

Bio-nanopatterning of surfaces is becoming a crucial technique with applications ranging from molecular and cell biology to medicine. Scanning probe microscopy (SPM) is one of the most useful tools for nanopatterning of flat surfaces. However, these patterns are usually built on homogeneous surfaces and require chemical functionalization to ensure specific affinity. Layered magnesium-aluminum hydroxide-silicates have already shown unique self-assembly properties on DNA molecules, due to their peculiar crystal chemistry based on alternating positive and negative crystal layers. However, patterns on these surfaces tend to be randomly organized. Here we show etching and oxidation at the nanometer scale of magnesium-aluminum hydroxide-silicates using the same SPM probe for the creation of organized nanopatterns. In particular, it is possible to produce three-dimensional structures in a reproducible way, with a depth resolution of 0.4 nm, lateral resolution of tens of nm, and a speed of about 10 µm s(-1). We report, as an example, the construction of an atomically flat charged pattern, designed to guide DNA deposition along predetermined directions without the need of any chemical functionalization of the surface.

Note: radiofrequency scanning probe microscopy using vertically oriented cantilevers.

We present a simple method, based on the modification of a widespread atomic force microscope, that allows the simultaneous acquisition of the sample topography and RF spectra at the nanoscale minimizing the parasitic capacitance of the cantilever. We used a microcantilever set with its long axis perpendicular to the specimen surface and connected to a vector network analyzer (RF range 100 kHz-8.5 GHz) to measure RF impedance signal variations at the cantilever apex-sample interface. The RF impedance signal was found highly sensitive to very short probe-to-sample distances (<50 nm) and to material properties at the interface.

Controlled positive and negative surface charge injection and erasure in a GaAs/AlGaAs based microdevice by scanning probe microscopy.

In this paper, we show that positive and negative charges can be injected into the surface of SiO(2)/Si(3)N(4)/SiO(2)/GaAs/AlGaAs heterostructure material by using a biased tip of a scanning probe microscope. Furthermore, the injected charges can be erased with the same tip once grounded, working in slow scan and contact mode. Surface potential measurements by quantitative analysis of Kelvin probe force microscopy after drawing and erasing charges at room temperature are presented and discussed.

Lipid oxidation deletes the nanodomain organization of artificial membranes.

Nanoscopic domains with different crystal structures have been induced in closed artificial membranes and have been directly imaged by atomic force microscopy at a spatial resolution better than 0.3 nm. These observations provide experimental evidence to the hydrophobic mismatching theory of lateral phase separation phenomena. Under oxidant conditions, the lipid-lipid assembly reorganizes into a new steady-state structure with disappearance of specific nanodomains. This finding may contribute to understanding the mechanism of peroxidative damage to membrane properties. In fact, alterations of specific modes of molecular conformation and packing may lead to perturbation of specific properties.

Bioactivity of chitosan in dentistry. Preliminary data on chitosan-based cements.

BACKGROUND: The chemical association of chitosan with inorganic salts, such as calcium phosphate, finds a promising application in dentistry as room-temperature self-hardening cement. We present the physical, chemical and crystallographic characterization of newly-developed cements made of 1) calcium-phosphate and a chitosan gel obtained by acetic acid treatment, and 2) calcium phosphate and a chitosan gel obtained by ascorbic acid treatment. Both cements are self-hardening at room temperature. METHODS: The cements were characterized by X-ray diffractography, scanning electron microscopy and fluorine-selective electrode analysis. RESULTS: The chitosan-hydroxyapatite cements had hardness comparable to spongy bone and above that of PMMA. CONCLUSIONS: The cements are promising for application in endodontics and restorative dentistry.

Calcium phosphates produced by physical methods in the treatment of dentin hypersensitivity.

BACKGROUND: The aim of this paper is to study the properties of innovative materials based on defective calcium phosphates produced by physical methods in the therapy of dentin hypersensitivity. METHODS: In particular, the effects of gels, aqueous solutions and toothpastes containing the above mentioned materials on dentinal permeability measured as dentin hydraulic conductance have been studied. The calcium phosphates have been characterized by X-ray powder diffraction (Rietveld analysis) and Fourier transform infra-red analysis. In addition, scanning electron microscopy has been performed to study the surface of dentin and enamel after treatment with the phosphates. In particular sound occlusal dentin, sound cervical dentin, carious occlusal dentin, sound buccal enamel and carious buccal enamel have been observed. RESULTS: The results have shown that these biocompatible materials can be produced with chemical and physical characteristics very similar to dentin and/or enamel. By forming a protective layer inside and outside the dentin tubuli, the calcium phosphates significantly reduce the dentinal hypersensitivity. CONCLUSIONS: These phosphates seem to be a promising material for clinical application.

Transformation reactions of partially gold-coated bioactive Ca-P glass granules investigated by analytical scanning electron microscopy.

In the present work, we have clarified the detail of the surface transformation reactions of bioactive calcium-phosphate (Ca-P) glass granules induced by in vivo implantation in rabbit dorsal muscle sites. To this aim we have compared the behaviour, during the same implantation, between the as-prepared and gold-coated only-on-one-side glass granules. The deposited gold layer enabled us to determine very precisely the initial position of the surface of the glass before the transformation took place. In addition, since the gold layer acts as a diffusion barrier, it allowed the study of the direction and the mechanism of crystal growth which occurred at the glass surface. Lapped and polished cross-sections of the samples were examined by backscattered electron (BSE) imaging and quantitative (with standards) x-ray energy dispersive spectroscopy (EDS) in a scanning electron microscope (SEM). The observations showed the presence of an interlayered structure. Quantitative EDS microanalysis performed by profiling the electron beam across the samples indicated the presence of hydrated calcium phosphate in the external layer, an inhomogeneous silica-rich gel-type layer in the middle layer, and an unaffected original Ca-P glass in the centre. From the comparison with those granules gold-coated on one side, we deduced that the hydrated calcium phosphate layer grew towards the interior of the granules at the expense of the starting glass. A simple model, based on the balance of the concentrations of the elements which have diffused in the different layers, is proposed to explain the contribution of the elements constituting the original glass to the formation of the different layers. This result agrees with the experimental data obtained from image analysis and the microstructural behaviour of this type of glass is discussed.

In vitro calcified tendon collagen: an atomic force and scanning electron microscopy investigation.

Atomic force microscopy (AFM), scanning electron microscopy and X-ray energy dispersive spectroscopy have been performed on decalcified turkey tendons submitted to in vitro calcification in order to investigate the morphology and the surface relationships between the inorganic phase and the collagen fibres during deposition and compare with those found for physiologically calcified samples. 'Tapping mode' AFM was used to reduce the vertical force applied to the samples, which were examined without any preparation. A further characterization has been carried out by means of X-ray diffraction, infrared absorption and chemical analyses. The observations indicate that the inorganic phase deposited on collagen fibres during in vitro calcification is poorly crystalline B carbonated apatite. The composition, structure and dimensions of apatitic crystallites, as well as their orientation with respect to collagen fibrils, are very similar to those characteristic of physiologically calcified tissues. However, the crystallites seem to be nucleated on the fibril surface, without appreciably affecting the molecular packing of collagen.

Importance of microanalysis in understanding mechanism of transformation in active glassy biomaterials.

This work deals with a method to study bioactive glassy materials used for repair of bone defects in order to understand the mechanism of the bioactivity and thus have its in vivo behavior optimized. The study of these materials takes into account their changes in morphology, by means of electron microscopy, after implantation in different animal models and evaluates the mechanism of the bioactivity through semi- and quantitative microanalytical evaluations in order to quantify the phenomenon of glass corrosion and the formation of a chemical bond with bone. Special preparations of glass granules before implantation were prepared to microanalytically study surfaces with different permeabilities.

Atomic force microscopy observations of acyl chains in phospholipids.

The potential use of atomic force microscopy (AFM) to image the mode of assembly and to measure the corresponding lattice parameters of model systems consisting of ordered aggregates of cardiolipin molecules has been investigated. An unprecedented resolution of about 0.2 nm has been achieved on suitably prepared specimens. This enables the orientational order and the positional correlations of the individual molecules in the lattice to be defined, and submolecular details, such as the acyl chains and the polar groups, to be imaged. The structural parameters derived from AFM have been compared with those obtained by transmission electron diffraction of the same specimen and found to be in excellent agreement. AFM turns out to be a powerful and probably a unique tool to reveal local phase variations in systems, such as biological membranes, that have non-homogeneous composition and organization.

[The characteristics and properties of calcium phosphates in biomaterial formulations used in dentistry. 1]. / Caratteristiche e proprietà di alcuni fosfati di calcio presenti nella formulazione di biomateriali impiegati in odontostomatologia. Nota 1.

In this work we present the features and the properties of natural and synthetic apatites, which we use in an experimental study of various commercial products composed by calcium phosphate. After having considered the general concepts of biomaterials and biocompatibility, we describe non biological tests used for the characterization of these products. Biomaterials used in this study are: reabsorbable Dac Blu, non reabsorbable Dac Blu, non reabsorbable atomized Dac Blu, non reabsorbable thin Dac Blu, reabsorbable Biocoral 450, Calcitite 2040-12, Orthogel, Apagen, BTF 65, Calcitite 4060-2 Osprogel, Bio-oss, Biostite, Osprovit, Merck Hydroxiapatite. Tests used are: X-ray fluorescence (XRF), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), thermogravimetric and thermodifferential analysis (TG, DTG, DTA), scanner-ing electron microscopy (SEM) morphological analysis. Last but not least, we underline the particular features of these tests whose interpretation allows a more precise definition of the bioactivity of a biocompatible material.

[The thermal analysis of mineral-based biomaterials used in dentistry. 5]. / Analisi termiche di biomateriali a base minerale utilizzati in odontostomatologia. Nota 5.

In this study the following commercial products were thermo-analysed (TG, DTG, DTA): reabsorbable Dac Blu, non reabsorbable Dac Blu, non reabsorbable atomized Dac Blu, non reabsorbable thin Dac Blu, reabsorbable Biocoral 450, non reabsorbable thin Dac Blu, reabsorbable Biocoral 450, Calcitite 2040-12, Orthogel, Apagen, BTF 65, Calcitite 4060-2, Osprogel, Bio-Oss, Biostite, Osprovit, Merck Hydroxiapatite. The apparatus utilized was a Setaram TAG 24, in a symmetrical set up, which gave the TG, DTG and DTA analysis simultaneously. The analysis of all the materials were performed in correct operative conditions. The thermo analyses gave useful information about the thermal stability of the samples. The TG analysis gave the data relative to the percentage of weight loss due to the volatile substances in the samples (H2Oass C, OH-, CO2). The DTG graph made it possible to establish the presence of carbonate apatite and/or hydroxyapatite or the absence of both the substances. Finally, DTA and DTG analyses allowed show the presence of various organic substances. These data were very useful to characterize the examined materials and confirmed the extreme importance and sensibility of this method.

[The X-ray fluorescence (XRF) spectrometry of biomaterials used in dentistry. 2]. / Analisi in spettrometria per fluorescenza di raggi X (XRF) di biomateriali utilizzati in odontostomatologia. Nota 2.

In this work we analyze the following biomaterial: biomaterials used in this study are: reabsorbable Dac Blu, non reabsorbable Dac Blu, non reabsorbable atomized Dac Blu, non reabsorbable thin Dac Blu, reabsorbable Biocoral 450, Calcitite 2040-12, Orthogel, Apagen, BTF 65, Calcitite 4060-2, Osprogel, Bio-Oss, Biostite, Osprovit, Merck Hydroxiapatite. The quantitative XRF analysis was performed by a Philips PW 1480 with Rh tube. This research, besides underlining the possibility of applying the XRF method to the analysis of biomaterials. This study, shows the facility and the rapidity in the preparation of samples and standards in the form of tablets to undergo the analysis: furthermore the study shows the possibility of verify the analysis on the same sample in the future, because the tablet if well conserved, does not deteriorate. We can also verify a good analytic accuracy both for the principal elements (Ca, P) and for trace elements. The analyses show a moderate variability in the Ca/P ratio in the hydroxylapatites, and a greater variability in the secondary and trace elements.

[X-ray powder diffraction (XRD) in the study of biomaterials used in dentistry. 3]. / La diffrattometria X (XRD) a polveri nello studio di biomateriali utilizzati in odontostomatologia. Nota 3.

This study considers X-ray power diffraction (XRD) applied to the study of mineral-based biomaterials used in odontostomatology. By means of this method the following materials were analysed: reabsorbable Dac Blu, non reabsorbable Dac Blu, non reabsorbable atomized Dac Blu, non reabsorbable fine Dac Blu, reabsorbable Biocoral 450, Calcitite 2040-12, Orthogel, Apagen, BTF 65, Calcitite 4060-2, Osprogel, Bio-oss, Biostite, Osprovit, Merck Hydroxyapatite. These analysis allow the identification of the crystalline phases, the study of the crystallinity and the crystal chemistry of the samples prepared as powder mixtures. This method permits the determination of the physical chemical and crystalline characteristics of these mineral based biomaterials formed by powders or transformable in powders. All of this information is indispensable for the evaluation of the functional biocompatibility of a biomaterial when its reaction in a biological environment is already known. This method has a great number of advantages against the traditional methods, marking with solid phases, it does not destroy the sample, it does not modify the physical or chemical characteristics and gives more information.

[The characterization by Fourier-transform infrared vibration spectroscopy of mineral-based biomaterials commonly used in dentistry. 4]. / Caratterizzazione mediante spettroscopia vibrazionale infrarossa a trasformata di Fourier (FTIR) di alcuni biomateriali a base minerale di uso comune in odontostomatologia. Nota 4.

In this study the following biomaterials were analysed by means of Fourier transform infrared vibrational spectroscopy (FTIR); they are: reabsorbable Dac Blu, non reabsorbable Dac Blu, non reabsorbable atomized Dac Blu, non reabsorbable fine Dac Blu, reabsorbable Biocoral 450, Calcitite 2040-12, Orthogel, Apagen, BTF 65, Calcitite 4060-2 Osprogel, Bio-Oss, Biostite, Osprovit, Merck Hydroxyapatite. The analysis was performed with a Jasco 5300-FTIR. At 64 readings for spectrum with a 2 cm-1 resolution. By means of this method it has been possible to perform a structural study at a molecular level of the commercial products mentioned before. The analysis showed which of the samples examined had lost the apatite OH- group during production. It also allowed the identification of some of the hydroxyapatites examined as carbonate apatites. Finally, it allowed the identification and quantification of the organic substances in the examined products.