Customized hybrid biomimetic hydroxyapatite scaffold for bone tissue regeneration.

Three-dimension (3D) scaffolds for bone tissue regeneration were produced combining three different phases: nanometric hydroxyapatite (HA) was synthesized by precipitation method and the crystals nucleation took place directly within collagen fibrils following a biologically inspired mineralization process; polycaprolactone was employed to give the material a 3D structure. The chemico-physical analysis carried out to test the material's properties and composition revealed a high similarity in composition and morphology with biologically mineralized collagen fibrils and a scaffold degradation pattern suitable for physiological processes. The micro- computerized tomography (micro-CT) showed 53.53% porosity and a 97.86% mean interconnected pores. Computer-aided design and computer-aided manufacturing (CAD-CAM) technology was used for molding the scaffold's volume (design/shape) and for guiding the surgical procedure (cutting guides). The custom made scaffolds were implanted in sheep mandible using prototyped surgical guides and customized bone plates. After three months healing, scanning electron microscopy (SEM) analysis of the explanted scaffold revealed a massive cell seeding of the scaffold, with cell infiltration within the scaffold's interconnected pores. The micro-CT of the explanted construct showed a good match between the scaffold and the adjacent host's bone, to shield the implant primary stability. Histology confirmed cell penetration and widely documented neoangiogenesis within the entire scaffold's volume. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 723-734, 2017.

Vibration absorption using non-dissipative complex attachments with impacts and parametric stiffness.

Studies on prototypical systems that consist of a set of complex attachments, coupled to a primary structure characterized by a single degree of freedom system, have shown that vibratory energy can be transported away from the primary through use of complex undamped resonators. Properties and use of these subsystems as by energy absorbers have also been proposed, particularly using attachments that consist of a large set of resonators. These ideas have been originally developed for linear systems and they provided insight into energy sharing phenomenon in large structures like ships, airplanes, and cars, where interior substructures interact with a master structure, e.g., the hull, the fuselage, or the car body. This paper examines the effects of nonlinearities that develop in the attachments, making them even more complex. Specifically, two different nonlinearities are considered: (1) Those generated by impacts that develop among the attached resonators, and (2) parametric effects produced by time-varying stiffness of the resonators. Both the impacts and the parametric effects improve the results obtained using linear oscillators in terms of inhibiting transported energy from returning to the primary structure. The results are indeed comparable with those obtained using linear oscillators but with special frequency distributions, as in the findings of some recent papers by the same authors. Numerically obtained results show how energy is confined among the attached oscillators.

Energy equipartition and frequency distribution in complex attachments.

As reported in several recent publications, an undamped simple oscillator with a complex attachment that consists of a set of undamped parallel resonators can exhibit unusual energy sharing properties. The conservative set of oscillators of the attachment can absorb nearly all the impulsive energy applied to the primary oscillator to which it is connected. The key factor in the ability of the attachment to absorb energy with near irreversibility correlates with the natural frequency distribution of the resonators within it. The reported results also show that a family of optimal frequency distributions can be determined on the basis of a variational approach, minimizing a certain functional related to the system response. The present paper establishes a link between these optimal frequency distributions and the energy equipartition principle: optimal frequency distributions are those that spread the injected energy as uniformly as possible over the degrees of freedom or over the modes of the system. Theoretical as well as numerical results presented support this point of view.

The thermal transformation of Man Made Vitreous Fibers (MMVF) and safe recycling as secondary raw materials (SRM).

This work describes the high temperature reaction sequence of commercial Man Made Vitreous Fibers (MMVF) Cerafiber, Superwool, Rock wool and Glass wool which may be used as substitute for asbestos in some industrial applications. Knowledge of the reaction path and transformation sequence is very important to assess whether carcinogenic crystalline phases are formed during devitrification, which may occur when used as insulators. In addition, knowledge about the nature of the phases formed at high temperature is mandatory to assess if thermally transformed MMVF can be safely recycled as secondary raw material (SRM). In this scenario, this study provides useful information for the optimization of the industrial annealing process aimed to attain a safe, recyclable product. The results of this work show that one of the high-temperature products of Cerafiber and Superwool is cristobalite which is classified as a carcinogenic. It was possible to define the temperature interval at which Cerafiber and Superwool fibers can be safely used as thermal insulators (e.g. insulators in tunnel and/or roller kilns, etc.). As cristobalite is formed in both synthetic fiber products at temperatures higher than 1200 degrees C, their use should be limited to devices operating at lower temperatures. Rock and Glass wool melt upon thermal treatment. As far as the industrial process of inertization is concerned, a maximum firing temperature of 1100 and 600 degrees C is required to melt Rock wool and Glass wool, respectively, with the high-temperature products that can be safely recycled as SRM. Recycling of these products in stoneware tile mixtures were subsequently attempted. The addition of 1-2 wt.% of the melts of Rock and Glass wool gave promising results in terms of viscous sintering reactions and resistance to staining with the only weak characteristic being the color properties of the fired bodies which tend to worsen.

Mitochondrial changes induced by natural and synthetic asbestos fibers: studies on isolated mitochondria.

Asbestos fibers, such as chrysotile and crocidolite, are known to have cytotoxic effects on different cell types. In vivo exposure to asbestos fibers can induce both fibrotic and malignant lung diseases , however, the mechanisms linking exposure to the subsequent development of the diseases are unknown. Numerous investigations suggest the involvement of reactive oxygen species (ROS). ROS are known to damage biological macromolecules including proteins, cell membrane lipids and nucleic acids; alterations of these essential cellular components can alter cell function and can drive the cell to neoplastic transformation or to cell death. Because the mitochondrial respiratory chain is an important source of ROS and RNS (reactive nitogen species) in the cells, we have investigated the effects of aqueous extracts of asbestos (natural and synthetic) fibers on some mitochondrial activities. Our data show that crocidolite fibers release substances in solution that may interfere directly with the mitochondrial cytochrome oxidase complex. Moreover, the calcium ions released from these fibers induce opening of the permeability transition pore of the inner membrane leading to a possible cytotoxic effect due to the release of apoptotic factors normally localized in the mitochondrial intermembrane space. In addition, crocidolite extracts enhance the mitochondrial production of ROS. No significant biochemical effects are exerted by chrysotile, either natural or synthetic, on isolated mitochondria. Nevertheless, all asbestos fibers tested induce morphological alterations visualized by transmission electron microscopy and morphometric analysis.

Silica xerogels as a delivery system for the controlled release of different molecular weight heparins.

In this work, we investigated a sol-gel derived silica matrix as a delivery system for the prolonged release of different molecular weight heparins, which allows the glycosaminoglicons to retain their whole biological activity. Several xerogels were obtained by embedding different molecular weight heparins into matrices prepared by using different amount of NH4OH as a catalyst during gel formation. Gel synthesis parameters, drug release properties, and xerogels surface area were evaluated. Unfractionated, low and oligo-molecular weight heparins were embedded into xerogels and the effect of the molecular weight on the release kinetics and the retained biological activity has been investigated. The results show that the surface area of the matrix is a determinant parameter affecting drug release kinetics. This structural feature can be modified by varying the catalyst tetraethoxysilane molar ratio used during the matrix synthesis. In most cases release kinetics fitted the Higuchi diffusive model and a lower diffusion rate was observed from silica matrices characterized by a smaller surface area. In the case of matrices with lower surface area, loaded with unfractionated heparin, zero order kinetics was observed. In this paper, we have defined a heparin release silica xerogel system and we have pointed out how modulation of its synthesis parameters allows adjusting the release of heparin according to therapeutic needs.

HA/alginate hybrid composites prepared through bio-inspired nucleation.

Poorly crystalline apatite has been directly nucleated on self-assembling alginate chains by neutralization synthesis to obtain a biomimetic artificial bone-like composite. It has been observed that in preparing HA/alginate composites, Ca2+ ions present on the apatitic surface cross-link the alginate chains to produce a material with different morphology and thermal stability, both functions of the HA/alginate weight ratio. In vitro tests were performed on different samples in terms of both the HA/alginate ratio and synthesis temperature. All the samples were cultured for seven days with MG63 osteoblast-like cells and then underwent morphological and biochemical analyses (MTT and ALP tests). Scaffolds showed a different solubility into the culture media, which was related to the temperature of synthesis and to the HA/alginate ratio. All our data confirm the ability of the tested materials to favour cell growth and to maintain their osteoblastic functionality, at least during the examined period.

Mitochondrial changes induced by natural and synthetic asbestos fibers: studies on isolated mitochondria.

Asbestos fibers, such as chrysotile and crocidolite, are known to have cytotoxic effects on different cell types. in vivo exposure to asbestos fibers can induce both fibrotic and malignant lung diseases , however, the mechanisms linking exposure to the subsequent development of the diseases are unknown. Numerous investigations suggest the involvement of reactive oxygen species (ROS). ROS are known to damage biological macromolecules including proteins, cell membrane lipids and nucleic acids; alterations of these essential cellular components can alter cell function and can drive the cell to neoplastic transformation or to cell death. Because the mitochondrial respiratory chain is an important source of ROS and RNS (reactive nitogen species) in the cells, we have investigated the effects of aqueous extracts of asbestos (natural and synthetic) fibers on some mitochondrial activities. Our data show that crocidolite fibers release substances in solution that may interfere directly with the mitochondrial cytochrome oxidase complex. Moreover, the calcium ions released from these fibers induce opening of the permeability transition pore of the inner membrane leading to a possible cytotoxic effect due to the release of apoptotic factors normally localized in the mitochondrial intermembrane space. In addition, crocidolite extracts enhance the mitochondrial production of ROS. No significant biochemical effects are exerted by chrysotile, either natural or synthetic, on isolated mitochondria. Nevertheless, all asbestos fibers tested induce morphological alterations visualized by transmission electron microscopy and morphometric analysis.

A new method for the detection of low levels of free fibres of chrysotile in contaminated soils by X-ray powder diffraction.

A new method for the determination of free fibres of chrysotile in contaminated soils is described. The detection limit of 0.5 wt per thousand is reached by an enrichment process of the asbestos fraction of the sample using a standard laboratory elutriator for sedimentation analysis. The analysis of the enriched fraction is performed by X-ray powder diffraction using a conventional instrument. The procedure can be successfully applied to several soils of different nature throughout thermal treatment and removal of possible interferences due to some matrix components. This method is straightforward, routinized and has been especially developed to fulfil the request of public and private institutions for an appropriate quantitative determination of chrysotile free fibres in contaminated soils.

Determination of low levels of free fibres of chrysotile in contaminated soils by X-ray diffraction and FTIR spectroscopy.

A new analytical method for the determination of low levels (0.01-1 wt%) of free fibres of chrysotile in contaminated clayey, sandy and sandy-organic soils is described. The detection limit of 0.01 wt% is reached with an enrichment of free fibres of chrysotile in the sample using a standard laboratory elutriator for sedimentation analysis. The chrysotile quantitative determination is performed both by X-ray powder diffraction, using the internal standard and reference intensity ratio methods, and by Fourier-transform infrared absorption spectroscopy. The procedure can be successfully applied to different soils after removal, by a thermal treatment, of the matrix components which can interfere. This straightforward method fulfils the request of public institutions and private companies for an appropriate quantitative determination of chrysotile-free fibres in contaminated soils.

Porous phosphate-gelatine composite as bone graft with drug delivery function.

The design and synthesis of porous phosphate-gelatine composite implant which mimicks the structure of natural bone and has drug delivery function is proposed. Gelatine reproducing the proteinaceous part of bone was cross-linked in order to modulate its solubility in the physiologic fluids. The kinetic of gelatine release from ceramic matrix was also evaluated as model of the release of any therapeutic compound which can be loaded into gelatine.

Stabilization of gelatin films by crosslinking with genipin.

The possibility to stabilize gelatin films by crosslinking with genipin was investigated through a mechanical, chemical and thermal characterization of samples treated with genipin solutions at different concentrations. The extent of crosslinking, evaluated as difference between the number of free epsilon -amino groups before and after crosslinking, increases as a function of genipin concentration up to about 85%. Simultaneously, the deformability of the films decreases whereas the Young's modulus E, increases. Furthermore, crosslinking provokes a significant reduction of the swelling in physiological solution, and enhances the thermal stability of the samples, as indicated by the results of the d.s.c. investigation. The data obtained from the films treated with genipin at concentrations higher than 0.67% are quite similar, and indicative of a good stabilizing effect of genipin. In spite of the small gelatin release (2%) observed after 1 month of storage in buffer solution, the mechanical, thermal and swelling properties of the films are very close to those previously obtained for glutaraldehyde crosslinked gelatin, and suggest that genipin, which is by far less cytotoxic, can be considered a valid alternative for crosslinking gelatin biomaterials.

Structural and morphological characterization of synthetic chrysotile single crystals.

Stoichiometric chrysotile single crystals have been synthesized as a unique phase by hydrothermal reaction under controlled conditions; the synthesized monocrystals show a cylinder-in-cylinder morphology and can be used as a reference sample with definite chemical composition to investigate the factors responsible of the chrysotile cytotoxicities and carcinogenicities.

Bonelike apatite growth on hydroxyapatite-gelatin sponges from simulated body fluid.

In vitro bioactivity of gelatin sponges and hydroxyapatite-enriched gelatin sponges was tested through evaluation of the variations in their composition and morphology after soaking in simulated body fluid (1.5) for periods up to 21 days at 37 degrees C. The presence of hydroxyapatite inside the sponges promotes the deposition of bonelike apatite crystals. The deposits are laid down as spherical aggregates, with mean diameters increasing from about 1-2 microm, after 4 days of soaking in simulated body fluid solution, up to about 3.5 microm in the samples soaked for 21 days. Simultaneously, the relative amount of inorganic phase increases up to about 56% wt, leading to a composite material with a composition quite close to that of bone tissue. The inorganic phase is a poor crystalline carbonated apatite similar to trabecular bone apatite.

Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking.

The mechanical, thermal, swelling and release properties of glutaraldehyde (GTA) crosslinked gelatin films have been investigated in order to verify the influence of GTA concentration on the stability of the films. Air-dried films were submitted to treatment with GTA solutions at concentrations ranging from 0.05 to 2.5 wt%. At the smallest GTA concentration, the crosslinking degree, determined by trinitrobenzensulfonic acid assay, amounts to about 60% and increases up to values near 100%, obtained with GTA concentrations > or = 1 wt%. Simultaneously, the deformability of the films decreases, whereas the stress at break, sigmab, and the Young's modulus, E, increase. A crosslinking degree of about 85%, obtained using 0.25% GTA, is enough to prevent gelatin release in buffer solution and to provoke a significant reduction of the swelling in physiological solution. Furthermore, crosslinking greatly affects the thermal stability of the samples, as indicated by the results of differential scanning calorimetry (d.s.c.) investigation carried out on wet and air-dried films. The data suggest that the use of GTA at low concentration, which is desiderable to prevent toxicity, allows to modulate the physico-chemical properties of gelatin films, in order to obtain stable materials with a wide range of possible biomedical applications.

Effect of sodium polyacrylate on the hydrolysis of octacalcium phosphate.

Octacalcium phosphate (OCP) hydrolysis into hydroxyapatite (HA) has been investigated in aqueous solutions at different concentrations of sodium polyacrylate (NaPA). In the absence of the polyelectrolyte, OCP undergoes a complete transformation into HA in 48 h. The hydrolysis is inhibited by the polymer, which is significantly adsorbed on the crystals, up to about 22 wt.%. A polymer concentration of 10(-2) mM is sufficient to cause a partial inhibition of OCP to HA transformation, which is completely hindered at higher concentrations. The small platelet-like crystals in the TEM images of partially converted OCP can display electron diffraction patterns characteristic either of OCP single crystals or of polycrystalline HA, whereas the much bigger plate-like crystals exhibit diffraction patterns characteristic of OCP single crystals. The polyelectrolyte adsorption on OCP crystals is accompanied by an increase of their mean length and by a significant reduction of the coherence length of the perfect crystalline domains along the c-axis direction. It is suggested that the carboxylate-rich polyelectrolyte is adsorbed on the hydrated layer of the OCP (100) face, thus inhibiting its in situ hydrolysis into HA.

Structural aspects of the calcification process of lower vertebrate collagen.

In order to investigate the structural relationship between inorganic phase and collagen fibrils in the calcified tissues of lower vertebrates we have carried out a wide and small angle X-ray diffraction investigation on carp scales and bone samples. The small angle patterns from decalcified bone and scales, as well as uncalcified tendon samples from carp are very similar to that of type I collagen from higher vertebrates. The D-axial period, 67 nm, is the same as that of higher vertebrate type I collagen, while the most significant difference is the relatively low intensity of the first order reflection, which is, however, the most intense. The relative intensity distributions of the meridional reflections recorded from fish bone and scales are in agreement with an electron density distribution according to a step function. The calculated step length is very close to the values previously reported for calcified tissues from higher vertebrates. The small angle reflections from calcified, as well as decalcified, scales display different directions of orientation, which could be in agreement with a plywood arrangement of collagen fibrils in successive sheets parallel to the plane of the scale.

Biomimetic growth of hydroxyapatite on gelatin films doped with sodium polyacrylate.

Gelatin films were used as biomimetic substrates for the nucleation of hydroxyapatite from simulated body fluid (SBF). Stretching and presence of sodium polyacrylate appear to be essential factors for the specific nucleation and growth of hydroxyapatite crystals inside the films. After soaking in 1.5SBF for periods longer than 4 days, all the films display a completely calcified surface. However, the spherical aggregates on the film surfaces do not give any X-ray diffraction effect and exhibit a low Ca/P molar ratio, typical of amorphous calcium phosphate. The ordered deposition of crystalline hydroxyapatite has been verified to take place only in stretched polyacrylate--gelatin films. The crystals grow as tablets about 2 microns thick among the gelatin layers, with their crystallographic c-axes preferentially oriented parallel to the direction of orientation of the collagen molecules, thus resembling the parallel orientation of apatitic crystals and collagen fibrils in calcified biological tissues.

Nanocrystals of magnesium and fluoride substituted hydroxyapatite.

Hydroxyapatite nanocrystals synthetized in the presence of different concentrations of magnesium and fluoride ions in solutions--1, 5 and 10 at.% have been submitted to a structural and chemical characterization. The syntheses were carried out in the presence of low molecular weight polyacrylic acid, which has been verified to inhibit hydroxyapatite crystallization. The polyelectrolyte is adsorbed into the crystals during the synthesis and provokes a reduction of the mean crystal sizes. The reduction is greater along the direction orthogonal to the c-axis, suggesting a preferential adsorption of the polyelectrolyte on the crystalline faces parallel to the c-axis. Both magnesium and fluoride can be incorporated into the hydroxyapatite structure. On the basis of the values of the lattice constants and of the magnesium relative content of the solid phase, it can be suggested that probably just a part of magnesium is substituted for calcium, the remainder being adsorbed on the crystal surface. However, magnesium destabilizes the apatitic structure favouring its thermal conversion into beta-tricalcium phosphate, and displays an inhibiting effect on the crystallization of hydroxyapatite. This last effect is enhanced by the simultaneous presence of polyacrylic acid. Fluoride substitution for hydroxyl ions into hydroxyapatite structure induces a slight increase of the crystal sizes along the c-axis direction. The data indicate that the experimental approach can be successfully used to prepare nanoapatite with crystallinity, crystal dimensions, composition, structure and stability very close to those characteristics of biological apatites.

Hydroxyapatite-gelatin films: a structural and mechanical characterization.

Composite films of gelatin and hydroxyapatite were prepared and characterized by mechanical tests, scanning electron microscopy and X-ray diffraction investigation. The mechanical properties of the films are greatly affected by the presence of hydroxyapatite and change as a function of inorganic phase content. On stretching, the long axis of the collagen molecular portions align parallel to the direction of deformation and the gelatin coarse layered structure becomes more evident and ordered. Furthermore, under deformation the inorganic crystals, which are embedded in the gelatin layers, seem to squeeze out in the interlayer spaces and assume a preferential orientation parallel to the force trajectories. Thus, as the inorganic phase stiffens the gelatin films, the macromolecular matrix distributes the stress promoting the preferential orientation of the apatitic crystals. The results indicate that this experimental approach can be used to prepare composites with anisotropic properties, which can be modulated through variation in composition and mechanical deformation in order to get biomaterials suitable to fulfill specific mechanical functions.