Valentina Aina

Zinc-containing bioactive glasses: surface reactivity and behaviour towards endothelial cells

This paper reports a physico-chemical study devoted to reactivity towards hydroxo-carbonate apatite (HCA) formation of bioactive glass 45S5 (H glass; commercially known as Bioglass) and of two preparations of zinc-doped 45S5-derived systems (HZ5, HZ20), immersed in Tris(hydroxymethyl)aminomethane (Tris) and Dulbeccos modified Eagles medium (DMEM) buffer solutions. The activity/toxicity of the glasses was also tested using endothelial cells (EC). Zn caused a drastic reduction in the overall leaching activity of glasses and, at high Zn concentration (HZ20), the formation of HCA on the glass surface was thoroughly inhibited. The presence of Zn also decreased the increment of pH after glass immersion in both Tris and DMEM solution. EC are known to be very sensitive to pH changes and, for this reason, the rapid increase in pH brought about by H glass dissolution is likely to affect cell adhesion and spreading, whereas the high zinc release from HZ20 causes a drastic reduction in cell proliferation after a long contact time (approximately 1 week). This study shows that only HZ5 glass containing 5 wt.% Zn presents at the same time: reduced solubility, bioactivity (monitored by HCA formation) and conditions allowing EC growth over a 6-day period.

Fluoride-containing bioactive glasses: surface reactivity in simulated body fluids solutions.

The issue of the contribution of the addition of F to glass bioactivity is not well resolved. This work reports on the surface reactivity in different solutions (DMEM and Tris) for some potentially bioactive glasses based on the composition of 45S5 glass, in which CaF(2) is substituted alternately for (part of) CaO and Na(2)O. The reactivity of F-containing glasses has been compared with that of the reference 45S5 system. The aim of this study is to explain in detail the mechanism of formation of an apatitic crystalline phase at the interface between the inorganic material and simulated biological media. A multi-technique investigation approach proposes a set of reactions involving Ca-carbonate formation, which are somewhat different from that formerly proposed by Hench for 45S5 bioactive glass, and which occur when a F-containing glass surface is in contact with a SBF. The usefulness of IR spectroscopy in recognizing the starting step of apatite (and/or FA) formation with respect to XRD technique is well established here.

Sr-containing hydroxyapatite: morphologies of HA crystals and bioactivity on osteoblast cells

A series of Sr-substituted hydroxyapatites (HA), of general formula Ca(10-x)Srx(PO4)6(OH)2, where x=2 and 4, were synthesized by solid state methods and characterized extensively. The reactivity of these materials in cell culture medium was evaluated, and the behavior towards MG-63 osteoblast cells (in terms of cytotoxicity and proliferation assays) was studied. Future in vivo studies will give further insights into the behavior of the materials. A paper by Lagergren et al. (1975), concerning Sr-substituted HA prepared by a solid state method, reports that the presence of Sr in the apatite composition strongly influences the apatite diffraction patterns. Zeglinsky et al. (2012) investigated Sr-substituted HA by ab initio methods and Rietveld analyses and reported changes in the HA unit cell volume and shape due to the Sr addition. To further clarify the role played by the addition of Sr on the physico-chemical properties of these materials we prepared Sr-substituted HA compositions by a solid state method, using different reagents, thermal treatments and a multi-technique approach. Our results indicated that the introduction of Sr at the levels considered here does influence the structure of HA. There is also evidence of a decrease in the crystallinity degree of the materials upon Sr addition. The introduction of increasing amounts of Sr into the HA composition causes a decrease in the specific surface area and an enrichment of Sr-apatite phase at the surface of the samples. Bioactivity tests show that the presence of Sr causes changes in particle size and/or morphology during soaking in MEM solution; on the contrary the morphology of pure HA does not change after 14 days of reaction. The presence of Sr, as Sr-substituted HA and SrCl2, in cultures of human MG-63 osteoblasts did not produce any cytotoxic effect. In fact, Sr-substituted HA increased the proliferation of osteoblast cells and enhanced cell differentiation: Sr in HA has a positive effect on MG-63 cells. In contrast, Sr ions alone, at the concentrations released by Sr-HA (1.21-3.24 ppm), influenced neither cell proliferation nor differentiation. Thus the positive effects of Sr in Sr-HA materials are probably due to the co-action of other ions such as Ca and P.

Properties of Zinc Releasing Surfaces for Clinical Applications

Two series of glasses of general formula (2-p) SiO2·1.1Na 2O·CaO·pP2O5·xZnO (p=0.10, 0.20; x=0.0, 0.16, 0.35, and 0.78) have been analyzed for physico-chemical surface features before and after contact with simulated body fluid, morphological characteristics, and osteoblast-like cells behavior when cultured on them. The resulted good cell adhesion and growth, along with nonsignificant changes of the focal contacts, allow the authors to indicate HZ5 and HP5Z5 glasses as the ones having optimal ratio of Zn/P to maintain acceptable cell behavior, comparable to the bioactive glass (Bioglass®) used as a control; results are also rationalized by means of three-dimensional models derived by molecular dynamic simulations, with decomposition and conversion rates optimized with respect to the parent Henchs Bioglass®.

Fluoride-containing bioactive glasses inhibit pentose phosphate oxidative pathway and glucose 6-phosphate dehydrogenase activity in human osteoblasts

Bioactive glasses such as Henchs 45S5 (Bioglass) have applications to tissue engineering as well as bone repair, and the insertion of fluoride in their composition has been proposed to enhance their bioactivity. In view of a potential clinical application, we investigated whether fluoride-containing glasses exert toxic effects on human MG-63 osteoblasts, and whether and how fluoride, which is released in the cell culture medium, might play a role in such cytotoxicity. A 24h incubation with 50 microg/ml (12.5 microg/cm(2)) of fluoride-containing bioactive glasses termed HCaCaF(2) (F content: 5, 10 and 15 mol.%) caused the release of lactate dehydrogenase in the extracellular medium (index of cytotoxicity), the accumulation of intracellular malonyldialdehyde (index of lipoperoxidation), and the increase of glutathione consumption. Furthermore, fluoride-containing glasses inhibited the pentose phosphate oxidative pathway and the glucose 6-phosphate dehydrogenase activity. These effects are ascribable to the fluoride content/release of glass powders, since they were mimicked by NaF solutions and were prevented by dimethyl sulfoxide and tempol (two radical scavengers), by superoxide dismutase (a superoxide scavenger), and by glutathione (the most important intracellular antioxidant molecule), but not by apocynin (an inhibitor of NADPH oxidase). The presence of fluoride-containing glasses and NaF caused also the generation of reactive oxygen species, which was prevented by superoxide dismutase and catalase. The data suggest that fluoride released from glasses is the cause of MG-63 cell oxidative damage and is independent of NADPH oxidase activation. Our data provide a new mechanism to explain F(-) ions toxicity: fluoride could trigger, at least in part, an oxidative stress via inhibition of the pentose phosphate oxidative pathway and, in particular, through the oxidative inhibition of glucose 6-phosphate dehydrogenase.

Engineered organic/inorganic hybrids for superhydrophobic coatings by wet and vapour procedures

Siloxane/oxide hybrids have attracted growing attention thanks to their ability to modulate the surface energy, wettability, or self-lubricity of a material. Here, we compare two functionalisation procedures (chemical vapour deposition and wet impregnation) on substrate films composed by preformed oxide particles. Three kinds of particles, characterised by different natures (SiO2 and TiO2) and average particle size, were studied to highlight possible effects related to the chemical and morphological state of the substrate surface. Morphological characterisations were carried out using dynamic light scattering and scanning electron microscopy, while the structure of the hydrophobing layer was studied by combining Fourier transform infrared spectroscopy and solid-state nuclear magnetic resonance. The degree of functionalisation and the features of the siloxane layer vary significantly among the adopted oxides and functionalisation methods. The wettability features of the different hybrids closely mirror the results of the spectroscopic characterisations, giving rise to either Lotus leaf or patch-wise hydrophobic systems depending on the adopted conditions.

The toxic effect of fluoride on MG-63 osteoblast cells is also dependent on the production of nitric oxide.

Some soda-lime-phospho-silicate glasses, such as Henchs Bioglass(®) 45S5, form bone-like apatite on their surface when bound to living bone. To improve their osteointegration for clinical purposes, the fluoride insertion in their structure has been proposed, but we recently showed that fluoride causes oxidative damage in human MG-63 osteoblasts, via inhibition of pentose phosphate oxidative pathway (PPP) and its key enzyme glucose 6-phosphate dehydrogenase (G6PD). In the same cells we have now investigated the role of nitric oxide (NO) in these effects. Fluoride-containing bioactive glasses and NaF caused, as expected, release of lactate dehydrogenase in the extracellular medium, accumulation of intracellular malonyldialdehyde, inhibition of PPP and G6PD: we have now observed that these effects were significantly reverted not only by superoxide dismutase (SOD) plus catalase (scavengers of reactive oxygen species), but also by N-monomethyl l-arginine (l-NMMA, a NOS inhibitor) and 2-phenyl-4,4,5,5,-tetramethylimidazoline-1oxyl 3-oxide (PTIO, a NO scavenger). Moreover the two highest concentrations of both fluoride-containing bioglasses and NaF caused increase of nitrite (a stable derivative of NO) levels in the culture supernatant, which was inhibited by l-NMMA, erythrocytes, PTIO and SOD/catalase, and increase of intracellular NO synthase (NOS) activity. The incubation with bioglasses or NaF increased also the phosphorylation of Ser(1177) in the endothelial NOS isoform. Furthermore, the NO donor spermine NONOate was able to inhibit G6PD activity in vitro, and this effect was partly reverted by PTIO. Therefore our results suggest that most cytotoxic effects of fluoride are mediated by the production of NO: reactive oxygen species are important, causing NOS phosphorylation. We also observed, for the first time, that Tempol, but not SOD/catalase, besides inhibiting the oxidative stress induced by fluoride, also scavenges fluoride ions. For this reason it is not a selective inhibitor of the oxidative effects of fluoride.

New Formulation of Functionalized Bioactive Glasses to Be Used as Carriers for the Development of pH-Stimuli Responsive Biomaterials for Bone Diseases

The aim of the present contribution is to prepare a functionalized bioactive glass potentially useful as prosthetic material, but also able to release organic molecules in response to a change of the pH environment. By this approach it is possible to develop devices which can be used for a triggered drug release in response to specific stimuli; this is an attractive research field, in order to avoid either systemic and/or local toxic effects of drugs. In particular, in the present paper we report data related to the development of a new formulation of bioactive glasses, their functionalization with organic molecules to obtain a pH-sensitive bond, their physicochemical characterization and in vitro bioactivity in simulated biological fluids (SBF), and organic molecule delivery tests at different pH. The glass functionalization, by means of a covalent reaction, allows us to produce a model of pH-responsive bioactive biomaterial: when it is exposed to specific pH changes, it can favor the release of the organic molecules directly at the target site. Cysteamine and 5-aminofluorescein are used as model molecules to simulate a drug. The materials, before and after the different functionalization steps and in vitro release tests at different pH, have been characterized by means of different experimental techniques such as X-ray powder diffraction (XRPD), Raman, FTIR and fluorescence spectroscopies, N2 adsorption, thermogravimetric (TGA) and elemental analysis.

Novel bio-conjugate materials: soybean peroxidase immobilized on bioactive glasses containing Au nanoparticles

In the field of implantation, the delivery and/or immobilization of biomolecules developing a specific action on bone mineralization has attracted great attention in the last few years. In fact, a wide spectrum of enzymes and proteins have been grafted with different methods onto/within implanted materials. Bioactive glasses and glass-ceramics, due to their tailorable properties in terms of chemical composition, reactivity, and easiness of manufacturing, represent good scaffolds for enzyme immobilization. These biomaterials are well known for their peculiar surface reactivity promoting, when contacted with real or simulated body fluids, the formation of an hydroxy-carbonate apatite layer. The aim of the present contribution has been to immobilize, via a covalent linkage, an enzyme on the glass surface through the formation of self-assembled monolayers (SAMs), in order to obtain a stable bio-conjugate useful as a material bio-implantable into the human body. The innovation of this study resides in the use of a new method of protein immobilization on the glass surface. Unlike other works, in which a preliminary silanization process has often been used, the introduction of gold nanoparticles (AuNPs) in the glass composition allowed us to exploit the easy SAMs formation process on the AuNPs dispersed in the bioactive glass matrix and, consequently, to immobilize an enzyme (soybean peroxidase, SBP, in the present case) on the SAMs. A thorough characterization of the materials, at different steps of the functionalization process, has been also reported, together with in vitro activity tests of immobilized SBP, compared with merely adsorbed SBP, and cytotoxicity tests using human osteoblast (MG-63) cells. Overall, a new bio-conjugate material, able to maintain its activity over time and to decrease the oxidative stress when in contact with MG-63 cells, has been obtained.

Gold-containing bioactive glasses: a solid-state synthesis to produce alternative biomaterials for bone implantations.

A new melted bioactive system containing gold nanoparticles (AuNPs) was prepared exploiting a post-synthesis thermal treatment that allows one to modify crystal phases and nature, shape and distribution of the gold species in the glass-ceramic matrix as evidenced by UV–visible spectroscopy, transmission electron microscopy and powder X-ray diffraction analysis. In human MG-63 osteoblasts the presence of Aun+ species caused an increase of lactate dehydrogenase leakage and malonyldialdehyde production, whereas Henchs Bioglass HAu-600-17 containing only AuNPs did not cause any effect. In addition, HAu-600-17 caused in vitro hydroxyapatite formation and an increase of specific surface area with a controlled release of gold species; this material is then suitable to be used as a model system for the controlled delivery of nanoparticles.