Calcite as a Precursor of Hydroxyapatite in the Early Biomineralization of Differentiating Human Bone-Marrow Mesenchymal Stem Cells.

Biomineralization is the process by which living organisms generate organized mineral crystals. In human cells, this phenomenon culminates with the formation of hydroxyapatite, which is a naturally occurring mineral form of calcium apatite. The mechanism that explains the genesis within the cell and the propagation of the mineral in the extracellular matrix still remains largely unexplained, and its characterization is highly controversial, especially in humans. In fact, up to now, biomineralization core knowledge has been provided by investigations on the advanced phases of this process. In this study, we characterize the contents of calcium depositions in human bone mesenchymal stem cells exposed to an osteogenic cocktail for 4 and 10 days using synchrotron-based cryo-soft-X-ray tomography and cryo-XANES microscopy. The reported results suggest crystalline calcite as a precursor of hydroxyapatite depositions within the cells in the biomineralization process. In particular, both calcite and hydroxyapatite were detected within the cell during the early phase of osteogenic differentiation. This striking finding may redefine most of the biomineralization models published so far, taking into account that they have been formulated using murine samples while studies in human cell lines are still scarce.

Mineral-organic hybrid nanotubes as highly sensitive solid state optical chemical sensors.

Hybrid materials represent one of the strategies of materials science for accomplishing complex functionalities hardly encompassed by single-component systems. The critical step in this approach is the mixing and/or bonding between the two different components, which must preserve the original characteristics of the materials or give rise to new functionalities originating from a proper and controlled interaction between the two components. Here, we demonstrate the use of the ionic self-assembly approach for fabricating functional nanomaterials comprising an inorganic matrix constituted by synthetic geomimetic chrysotile nanotubes and an organic superficial layer of a free-base porphyrin. The resulting hybrid nanomaterial can be processed as colloidal solution and as thin solid film. In both phases, the hybrid shows a bright red fluorescence under UV-blue excitation at ca. 400 nm. This fluorescence exhibits decreasing intensity with decreasing pH, as a result of the porphyrin J-type aggregation strongly catalyzed by the mineral surface. Simultaneously, the aggregation induces a neat color change from red to green, serving as a fast direct visual test of pH variations. These results open the route for the utilization of bio-compatible and inert mineral nanomaterials with strong adsorbing properties as efficient and cost-effective solid state vectors for functional molecules.

Electrospun nanostructured fibers of collagen-biomimetic apatite on titanium alloy.

Titanium and its alloys are currently the mainly used materials to manufacture orthopaedic implants due to their excellent mechanical properties and corrosion resistance. Although these materials are bioinert, the improvement of biological properties (e.g., bone implant contact) can be obtained by the application of a material that mimics the bone extracellular matrix. To this aim, this work describes a new method to produce nanostructured collagen-apatite composites on titanium alloy substrate, by combining electrospinning and biomimetic mineralization. The characterization results showed that the obtained mineralized scaffolds have morphological, structural, and chemical compositional features similar to natural bone extracellular matrix. Finally, the topographic distribution of the chemical composition in the mineralized matrix evaluated by Fourier Transform Infrared microspectroscopy demonstrated that the apatite nanocrystals cover the collagen fibers assembled by the electrospinning.

The iron-related molecular toxicity mechanism of synthetic asbestos nanofibres: a model study for high-aspect-ratio nanoparticles.

Asbestos shares with carbon nanotubes some morphological and physico-chemical features. An asbestos-like behaviour has been recently reported by some authors, though the mechanism of toxicity may be very different. To identify at the atomic level the source of toxicity in asbestos, the effect of progressive iron loading on a synthetic iron-free model nanofibre previously found non-toxic in cellular tests was studied. A set of five synthetic chrysotile nanofibres [(Mg,Fe)3(Si2O5)(OH)4] has been prepared with Fe ranging from 0 to 1.78 wt %. The relationship between fibre-induced free-radical generation and the physico-chemical characteristics of iron active sites was investigated with spin-trapping techniques on an aqueous suspension of the fibres and Mössbauer and EPR spectroscopies on the solids, respectively. The fully iron-free fibre was inert, whereas radical activity arose with even the smallest amount of iron. Surprisingly, such activity decreased upon increasing iron loading. Mössbauer and EPR revealed isolated iron ions in octahedral sites that undergo both axial and rhombic distortion and the occurrence of aggregated iron ions and/or extra-framework clustering. The isolated ions largely prevailed at the lowest loadings. Upon increasing the loading, the amount of isolated iron was reduced and the aggregation increased. A linear relationship between the formation of carbon-centred radicals and the amount of rhombic-distorted isolated iron sites was found. Even the smallest iron contamination imparts radical reactivity, hence toxicity, to any chrysotile outcrop, thereby discouraging the search for non-toxic chrysotile. The use of model solids that only differ in one property at a time appears to be the most successful approach for a molecular understanding of the physico-chemical determinants of toxicity. Such findings could also be useful in the design of safer nanofibres.

Spectroscopic characterization of Fe-doped synthetic chrysotile by EPR, DRS and magnetic susceptibility measurements.

Fe-doped synthetic geomimetic chrysotile nanocrystals represent a reference standard to investigate the health hazard associated with asbestos fibers and constitute interesting inorganic nanotubes for specific technological applications in light harvesting systems, optoelectronics and photonics. As the fiber toxicity is catalyzed by iron ions in specific crystallographic sites and the mechanical behaviour of synthetic chrysotile nanotubes is strongly affected by the iron doping extent, the characterization of Fe substitution to Mg and/or Si sites in the chrysotile structure appears highly important. By EPR, DRS spectroscopic analyses and magnetic investigations, Mg and/or Si ion replacement by Fe(3+) in a synthetic geomimetic chrysotile structure has been investigated. The results highlight that, as a function of the Fe doping extent and of the Fe doping process, iron can replace both Mg and Si sites. The contemporary iron substitution into the octahedral and tetrahedral sheets is associated with the presence of both of isolated Fe(3+) centres in high-spin 3d(5) configuration (S = 5/2, (6)A(1)((6)S)) in O(h) and T(d) symmetry and of intra-lattice clustered species. Increasing the Fe doping extent increases the concentration of aggregated species, while magnetic susceptibility confirms a paramagnetic anisotropy. The results allow to define the opportunity of using or not metallic Fe during the synthesis to obtain doped chrysotile nanocrystals with tailored morphological and structural properties suitable as a reference to study asbestos toxicity and apt to prepare new inorganic nanotubes and quantum wires for innovative technological applications.

Self-organizing functional materials via ionic self assembly: porphyrins H- and J-aggregates on synthetic chrysotile nanotubes.

Positively charged, synthetic chrysotile nanotubes act as inorganic tectons supporting H- and J-type aggregates of anionic porphyrins with markedly different optical properties, making these nanohybrid materials of interest for application in nanotechnology.

Ambient monitoring of asbestos in selected Italian living areas.

This paper presents the results of an intensive monitoring activity of the particulate, fall-out and soil of selected living areas in Italy with the aim to detect the asbestos concentration in air and subsequent risk of exposure for the population in ambient living environments, and to assess the nature of the other mineral phases composing the particulate matrix. Some areas were sorted out because of the presence of asbestos containing materials on site whereas others were used as blank spots in the attempt to detect the background environmental concentration of asbestos in air. Because the concentration of asbestos in ambient environments is presumably very low, and it is well known that conventional low-medium flow sampling systems with filters of small diameter (25mm) may collect only a very small fraction of particulate over a short period, for the first time here, an intense monitoring activity was conducted with a high flow sampling system. The high flow system requires the use of large cellulose filters with the advantage that, increasing the amount of collected dust, the probability to collect asbestos fibers increases. Both the protocol of monitoring and analysis are novel and prompted by the need to increase the sensitivity towards the small number of expected fibers. With this goal, the collection of fall-out samples (the particulate falling into a collector filled with distilled water during the monitoring shift) and soil samples was also accomplished. The analytical protocol of the matrix particulate included preliminary X-ray powder diffraction (XRPD), optical microscopy and quantitative electron microscopy (SEM and TEM). Correlations with climatic trends and PM10 concentration data were also attempted. The surprising outcome of this work is that, despite the nature of the investigated site, the amount of dispersed asbestos fibers is very low and invariably lower than the theoretical method detection limits of the SEM and TEM techniques for identification and counting of asbestos fibers. The results are compared to the literature data worldwide and an updated model for asbestos fibers dispersion in ambient environments is proposed.

Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages.

PURPOSE: The use of bacteriophages represents a valid alternative to conventional antimicrobial treatments, overcoming the widespread bacterial antibiotic resistance phenomenon. In this work, we evaluated whether biomimetic hydroxyapatite (HA) nanocrystals are able to enhance some properties of bacteriophages. The final goal of this study was to demonstrate that biomimetic HA nanocrystals can be used for bacteriophage delivery in the context of bacterial infections, and contribute - at the same time - to enhance some of the biological properties of the same bacteriophages such as stability, preservation, antimicrobial activity, and so on. MATERIALS AND METHODS: Phage isolation and characterization were carried out by using Mitomycin C and following double-layer agar technique. The biomimetic HA water suspension was synthesized in order to obtain nanocrystals with plate-like morphology and nanometric dimensions. The interaction of phages with the HA was investigated by dynamic light scattering and Zeta potential analyses. The cytotoxicity and intracellular killing activities of the phage-HA complex were evaluated in human hepatocellular carcinoma HepG2 cells. The bacterial inhibition capacity of the complex was assessed on chicken minced meat samples infected with Salmonella Rissen. RESULTS: Our data highlighted that the biomimetic HA nanocrystal-bacteriophage complex was more stable and more effective than phages alone in all tested experimental conditions. CONCLUSION: Our results evidenced the important contribution of biomimetic HA nanocrystals: they act as an excellent carrier for bacteriophage delivery and enhance its biological characteristics. This study confirmed the significant role of the mineral HA when it is complexed with biological entities like bacteriophages, as it has been shown for molecules such as lactoferrin.

The role of biomimetism in developing nanostructured inorganic matrices for drug delivery.

BACKGROUND: Biomimetism of synthetic biomaterials can be carried out at different levels, such as composition, structure, morphology, bulk and surface chemical-physical properties. Biomaterials can be turned into biomimetic imprinting of all these characteristics in order not only to optimise their interaction with biological tissues, but also to mimic biogenic materials in their functionalities. OBJECTIVE: This review outlines the biomimetic chemical-physical properties of inorganic matrices in controlling drug release. METHODS: This review is restricted to phosphates and silica among inorganic biomaterials proposed as drug delivery vehicles. CONCLUSION: By mimicking nature, we can design and synthesise inorganic smart materials that are reactive towards biological tissues and can release bioactive molecules by a kinetic that is controlled not only by the matrix tailored chemical-physical properties, but also by the response to stimuli induced by physiological or pathological processes.

Adsorption of human serum albumin on the chrysotile surface: a molecular dynamics and spectroscopic investigation.

The human serum albumin (HSA) secondary structure modifications induced by the chrysotile surface have been investigated via computational molecular dynamics (MD) and experimental infrared spectroscopy (FTIR) on synthetic chrysotile nanocrystals coated with different amount of HSA. MD simulations, conducted by placing various albumin subdomains close to the fixed chrysotile surface, show an initial adsorption phase, accompanied by local rearrangements of the albumin motifs in contact with the chrysotile layer. Next, large-scale rearrangements follow with consequent secondary structure modifications. Gaussian curve fitting of the FTIR spectra obtained for HSA-coated synthetic chrysotile nanocrystals has allowed the quantification of HSA structural modifications as a function of the amount of protein adsorbed. The experimental results support the atomistic computer simulations providing a realistic description of the adsorption of plasma proteins onto chrysotile and unravelling a key step in the understanding of asbestos toxicity.

Durable Self-Cleaning Coatings for Architectural Surfaces by Incorporation of TiO2 Nano-Particles into Hydroxyapatite Films.

To prevent soiling of marble exposed outdoors, the use of TiO2 nano-particles has been proposed in the literature by two main routes, both raising durability issues: (i) direct application to marble surface, with the risk of particle leaching by rainfall; (ii) particle incorporation into inorganic or organic coatings, with the risk of organic coating degradation catalyzed by TiO2 photoactivity. Here, we investigated the combination of nano-TiO2 and hydroxyapatite (HAP), previously developed for marble protection against dissolution in rain and mechanical consolidation. HAP-TiO2 combination was investigated by two routes: (i) sequential application of HAP followed by nano-TiO2 ("H+T"); (ii) simultaneous application by introducing nano-TiO2 into the phosphate solution used to form HAP ("HT"). The self-cleaning ability was evaluated before and after prolonged exposure to simulated rain. "H+T" and "HT" coatings exhibited much better resistance to nano-TiO2 leaching by rain, compared to TiO2 alone. In "H+T" samples, TiO2 nano-particles adhere better to HAP (having flower-like morphology and high specific surface area) than to marble. In "HT" samples, thanks to chemical bonds between nano-TiO2 and HAP, the particles are firmly incorporated in the HAP coating, which protects them from leaching by rain, without diminishing their photoactivity and without being degraded by them.

Synthetic chrysotile nanocrystals as a reference standard to investigate surface-induced serum albumin structural modifications.

Geoinspired synthetic chrysotile, which represents an ideal asbestos reference standard, has been utilized to investigate homomolecular exchange of bovine serum albumin (BSA), the major plasma protein, between the adsorbed and dissolved state at the interface between asbestos fibers and biological medium. FTIR spectroscopy has been used to quantify BSA structural modifications due to surface adhesion on chrysotile fibers as a function of the surface coating extent. Circular dichroism spectroscopy has been used to investigate the adsorption/desorption equilibrium through analysis of the BSA structural perturbations after protein desorption from chrysotile surface. Data results show clearly that in the solid state BSA modifications are driven by surface interaction with the substrate, following a bimodal adsorption evidenced by two different binding constants. On the other hand, BSA desorbed in solution is able to rearrange, in the lack of substrate, although keeping irreversible modifications with respect to the native species. The lack of regaining its native structure certainly affects albumin interaction with biological environment. The present investigation on the stoichiometric synthetic geoinspired chrysotile nanocrystals is the first approach toward a deeper attempt to use standard synthetic chrysotile reference samples in mimicking the behavior of asbestos fibers and allows to better understand their interaction with a biological environment.

Hydroxyapatite nanocrystals as a smart, pH sensitive, delivery system for kiteplatin.

Hydroxyapatite (HA) nanocrystals are important inorganic constituents of biological hard tissues in vertebrates and have been proposed as a bone substitute or a coating material for prostheses in biomedicine. Hydroxyapatite is also amenable for its capacity to bind to a great variety of biomolecules and therapeutic agents. As drug carriers, apatite nanoparticles also have the advantage of pH dependent solubility and low toxicity. Thus HA nanoparticles are negligibly soluble at physiological pH but their dissolution is accelerated at lower pH such as that typically found in the vicinity of tumors. In the present study we have investigated the adsorption on and the release from biomimetic HA nanoparticles of two platinum derivatives of cis-1,4-diaminocyclohexane ([PtX2(cis-1,4-DACH)], X2 = Cl2 (1) and 1,1-cyclobutanedicarboxylate (CBDCA, 2)). The first of the two compounds proved to be active against colon cancer cells also resistant to oxaliplatin. The release has been investigated as a function of pH to mimic the different physiological environments of healthy tissues and tumors, and the in vitro cytotoxicity of the releasates from the HA matrices has been assessed against various human cancer cell lines. The results fully confirmed the potential of 1-loaded HA nanoparticles as bone-specific drug delivery devices.

Lactoferrin Adsorbed onto Biomimetic Hydroxyapatite Nanocrystals Controlling - In Vivo - the Helicobacter pylori Infection.

BACKGROUND: The resistance of Helicobacter pylori to the antibiotic therapy poses the problem to discover new therapeutic approaches. Recently it has been stated that antibacterial, immunomodulatory, and antioxidant properties of lactoferrin are increased when this protein is surface-linked to biomimetic hydroxyapatite nanocrystals. OBJECTIVE: Based on these knowledge, the aim of the study was to investigate the efficacy of lactoferrin delivered by biomimetic hydroxyapatite nanoparticles with cell free supernatant from probiotic Lactobacillus paracasei as an alternative therapy against Helicobacter pylori infection. METHODS: Antibacterial and antinflammatory properties, humoral antibody induction, histopathological analysis and absence of side effects were evaluated in both in vitro and in vivo studies. RESULTS: The tests carried out have been demonstrated better performance of lactoferrin delivered by biomimetic hydroxyapatite nanoparticles combined with cell free supernatant from probiotic Lactobacillus paracasei compared to both lactoferrin and probiotic alone or pooled. CONCLUSION: These findings indicate the effectiveness and safety of our proposed therapy as alternative treatment for Helicobacter pylori infection.

Adsorption of the cis-[Pt(NH3)2(P2O7)](2-) (phosphaplatin) on hydroxyapatite nanocrystals as a smart way to selectively release activated cis-[Pt(NH3)2Cl2] (cisplatin) in tumor tissues.

The relevant adsorption of cis-[Pt(NH3)2(P2O7)](2-) (phosphaplatin) on hydroxyapatite nanocrystals (nHAP) was observed and studied in water suspension. Phosphaplatin cytotoxicity, which is very low for HeLa, MCF-7 and HS-5 cell lines could be enhanced, reaching that of cisplatin, by interaction with solid nHAP. This effect stems from nHAP ability to catalyze the phosphaplatin hydrolysis, producing the same hydrolytic species responsible for cisplatin antitumor activity.

Adsorption of bovine serum albumin onto synthetic Fe-doped geomimetic chrysotile.

Synthetic stoichiometric and Fe-doped geomimetic chrysotile nanocrystals represent a reference standard to investigate the health hazard associated with mineral asbestos fibres. Experimental evidence suggests that the generation of reactive oxygen species and other radicals, catalysed by iron ions at the fibre surface, plays an important role in asbestos-induced cytotoxicity and genotoxicity. In this study, structural modification of bovine serum albumin (BSA) adsorbed onto synthetic chrysotile doped with different amounts of Fe has been investigated by Fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA) and analytical pyrolysis coupled with gas chromatography-mass spectrometry. FT-IR data evidenced a marked increase in disordered structures like random coil and ß-turn of BSA-nanocrystal adduct with 0.52 wt% of Fe doped. The TGA profile of the BSA revealed that its interaction with the synthetic chrysotile surface was strongly affected by the substitution of Fe into the chrysotile structure. The 2,5-diketopiperazine yields, formed upon thermal degradation of the polypeptide chain (pyrolysis-gas chromatography), changed when the BSA was adsorbed on the nanofibres. In general, results suggested that minute amount (less than 1 wt%) of Fe doping in chrysotile affected the protein-nanofibre interactions, supporting the role that this element may play in asbestos toxicity. The catalytic role of iron and the consequent unfolding of protein due to the structural surface modification of nanofibres were also evaluated.

Metalated nucleotide chemisorption on hydroxyapatite.

The experiments here reported evidence on the importance of the residual charge of a nucleotide derivative, for the adsorption on nHAP (hydroxyapatite nanocrystals), in water solution. We found that the simple presence of phosphates on the nucleotide derivative does not guarantee adsorption on nHAP. On the other hand, we demonstrated that a cationic or neutral charge on a nucleotide derivative produces a strongly reduced chemical adsorption (chemisorption) whereas, in the presence of a net negative charge, relevant adsorption on nHAP is observed. The number of phosphates can only modulate the adsorption efficiency of a molecule provided that this latter bears an overall negative charge. The neutral zwitterionic nucleotide Pt(II) complexes, bearing negatively charged phosphates, are unable to give stable chemisorption. Previous considerations are important to model the binding ability of phosphate bearing nucleotide derivatives or molecules on hydroxyapatite. The findings reported in the present paper could be relevant in bone tissue targeting or nHAP mediated drug delivery.

Biologically inspired synthesis of bone-like composite: self-assembled collagen fibers/hydroxyapatite nanocrystals.

Replacement of bone tissue by graft materials and products of tissue engineering having composition, structure, and biological features that mimic natural tissue is a goal to be pursued. A biomimetic synthesis was performed to prepare new bone-like composites constituted of hydroxyapatite nanocrystals and self-assembled type I collagen fibers. We used a biological inspired approach that proved that the biological systems stored and processed information at the molecular level. Two different methodologies were used: dispersion of synthetic hydroxyapatite in telopeptides free collagen molecules solution and direct nucleation of hydroxyapatite into reconstituted collagen fibers during their assembling. The different preparation techniques were experimented then the composites thoroughly characterized and compared. Composite obtained by direct nucleation showed an intimated interaction of the inorganic and proteic components, which modified the apatitic phase and made its composition, morphology and structure similar to the mineral component of natural bone.

Remineralization and repair of enamel surface by biomimetic Zn-carbonate hydroxyapatite containing toothpaste: a comparative in vivo study.

Consumption of acidic foods and drinks and other factors that cause enamel wear are responsible for the daily enamel loss and degradation. Use of some toothpastes that have been showed to possess different properties of remineralisation and/or repair of the enamel surface may help to protect tooth enamel. The aim of this study was to evaluate whether the use of toothpaste containing Zn-carbonate hydroxyapatite (CHA) nanostructured microcrystals may exert remineralization/repair effects of the enamel surface. Two groups of patients, aged between 18 and 75 years, used a Zn-CHA nanocrystals-based toothpaste (experimental group) and a potassium nitrate/sodium fluoride toothpaste (active control group) for 8 weeks. At the end of this period, extractions were performed in five subjects per study group. Negative controls consisted of two subjects treated with non-specified fluoride toothpaste. Teeth were processed for morphological and chemical-physic superficial characterizations by means of Scanning Electronic Microscopy with Elementary analysis, X-Ray Diffraction analysis and Infrared analysis. In this study, the use of a Zn-CHA nanocrystals toothpaste led to a remineralization/repair of the enamel surface, by deposition of a hydroxyapatite-rich coating. On the other hand, the use of both a nitrate potassium/sodium fluoride and non-specified fluoride toothpastes did not appreciably change the enamel surface. In conclusion, this study demonstrates that the toothpaste containing Zn-CHA nanostructured microcrystals, differently from nitrate potassium/sodium fluoride and non-specified fluoride toothpastes, may promote enamel superficial repair by means of the formation of a protective biomimetic CHA coating.

Biological activity of lactoferrin-functionalized biomimetic hydroxyapatite nanocrystals.

The emergence of bacterial strains resistant to antibiotics is a general public health problem. Progress in developing new molecules with antimicrobial properties has been made. In this study, we evaluated the biological activity of a hybrid nanocomposite composed of synthetic biomimetic hydroxyapatite surface-functionalized by lactoferrin (LF-HA). We evaluated the antimicrobial, anti-inflammatory, and antioxidant properties of LF-HA and found that the composite was active against both Gram-positive and Gram-negative bacteria, and that it modulated proinflammatory and anti-inflammatory responses and enhanced antioxidant properties as compared with LF alone. These results indicate the possibility of using LF-HA as an antimicrobial system and biomimetic hydroxyapatite as a candidate for innovative biomedical applications.