Gianluca Malavasi

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.

Substitutions of cerium, gallium and zinc in ordered mesoporous bioactive glasses.

Ordered mesoporous glasses based on the 80% SiO(2)-15% CaO-5% P(2)O(5) system including up to 3.5% Ce(2)O(3), 3.5% Ga(2)O(3) or 7.0% ZnO (in mol.%) were synthesized by the evaporation-induced self-assembly process using Pluronic® 123 as a surfactant. An ordered hexagonal mesophase was observed in both the unsubstituted glass (denoted in this paper as B: blank) and glasses containing <0.4% of substituent by X-ray diffraction, transmission electron microscopy and electron diffraction. The increase in the amount of substituent led to a decrease in the mesopore order. B glass exhibited good textural properties: S(BET)=515m(2)g(-1), D(P)=4.7nm and V(P)=0.58cm(3)g(-1). With the inclusion of cerium, gallium and zinc oxides the textural properties decreased, but remained in amounts useful for clinical applications. Zinc-containing samples showed the highest decrement in the textural properties. Substituted glasses exhibited a quick in vitro bioactive response except when the ZnO content was over 0.4%. Taking into account the ordered mesoporosity, the quick in vitro bioactive response and the added values of the substituents, this new family of glasses are promising candidates for applications in bone tissue engineering.

Elucidation of the structural role of fluorine in potentially bioactive glasses by experimental and computational investigation.

Glasses belonging to the Na(2)O-CaO-P(2)O(5)-SiO(2) system and modified by CaF(2) substitution for CaO and Na(2)O alternatively, were synthesized and characterized experimentally and computationally. The results of molecular dynamics simulations show that fluorine is almost exclusively bonded to modifier cations (Ca and Na) with coordination number close to 4. A similar mean coordination number value is found in the crystal phases obtained by means of thermal treatment at fixed temperature. Addition of fluorine increases the polymerization of silicate tetrahedra by removing modifiers from the siliceous matrix. No appreciable amount of Si-F bonds are detected.

Synthesis and characterization of cerium-doped glasses and in vitro evaluation of bioactivity

Abstract The results of preparation, characterization and in vitro bioactivity evaluation of phosphosilicate glasses based on Bioglass® 45S5 (SiO2 45; Na2O 24.5; CaO 24.5; P2O5 6 wt%) doped during melting with (1.5–13.5 wt%) cerium dioxide (CeO2), has been reported. The choice of cerium was related to its low toxicity associated with bacteriostatic properties; cerium-doped bioactive glasses could be useful when implantation concerns local infected areas. The maximum value that permitted forming a homogeneous glass was 13.5 wt% and enabled us to get a better insight into CeO2 effect on the chemical behaviour of glasses. The as-quenched glasses were characterized by means of magnetic and spectroscopic measurements that revealed the prevailing presence of cerium (III). The bioactivity of the glasses was tested by soaking them in a simulated body fluid at 37 °C, under continuous stirring. ICP measurements were carried out for ion concentration determinations and the solution/glass interface was investigated by scanning electron microscopy equipped with energy dispersive analyzer (SEM/EDS technique) to check morphological modifications; the solids were investigated by means of X-ray powder diffraction and IR techniques. The results indicated that at low cerium content the glass degradation and repolymerization gave rise to an internal silicon-based layer and an external calcium-phosphate-based layer formed on the glass surface; high cerium content retards the glass degradation and gave rise to cerium–phosphate layer instead of calcium one. The cerium was never found in solution. For the reacted glasses, in the external layer, at 10% and 13.5% CeO2 contents the molar ratio Ca/P were ≈1 and 0.8 respectively and the molar ratio Ce/P was ≈0.3 in both cases. On the surface of the glasses with the highest cerium content it could distinguish new regular aggregates mainly formed by cerium and phosphate (Ce/P≈3.5, Ca/P≈0.6). In the cerium-free glass and with low cerium content (1.5 wt%) the calculated Ca/P ratio was 1.67, as calculated for pure hydroxyapatite. At low cerium content (BG-1.5 Ce) the glass behaviour was strictly similar to that of Bioglass® 45S5.

In vitro and in vivo behaviour of zinc-doped phosphosilicate glasses.

The aim of this work was to study the behaviour of zinc-doped phosphosilicate glasses based on Bioglass 45S5. In vitro (in simulated body fluid), the reactivity was analysed by means of inductively coupled plasma spectrometry, environmental scanning electron microscopy-energy-dispersive spectroscopy (ESEM-EDS) and X-ray diffraction. In vivo (a rat implanted with glass), the reactivity and the tissue behaviour were analysed by conventional histology, histochemistry, microradiography and ESEM-EDS. The in vivo behaviour matches that in vitro perfectly; they show comparable glass degradation processes and rates, ruled by the amount of zinc in the glass. The reaction mechanism for the formation of a polymerized silica layer superimposed with a peripheral calcium phosphate layer is clearly substantiated by ESEM-EDS investigations. The crystallization of a biologically active hydroxyapatite (HA) layer is observed in both cases; the in vitro experiment shows the presence of HA after 4 days.

Mesoporous bioactive scaffolds prepared with cerium-, gallium- and zinc-containing glasses

Mesoporous bioactive glass scaffolds (MBG_Scs), based on 80% SiO(2)-15% CaO-5% P(2)O(5) (in mol.%) mesoporous sol-gel glasses substituted with Ce(2)O(3), Ga(2)O(3) (both 0.2% or 1.0%) and ZnO (0.4% or 2.0%), were synthesized by combination of evaporation-induced self-assembly and rapid prototyping techniques. Cerium, gallium and zinc trace elements were selected because of their inherent beneficial biological properties. Fabricated scaffolds were characterized and compared with unsubstituted scaffold (B_Sc). All of them contained well interconnected ultralarge pores (pores >400 μm) ideal for vascular ingrowth and proliferation of cells. Macropores of size 100-400 μm were present inside the scaffolds. In addition, low-angle X-ray diffraction showed that B_Sc and scaffolds with substituent contents up to 0.4% exhibited ordered mesoporosity useful for hosting molecules with biological activity. The textural properties of B_Sc were a surface area of 398 m(2) g(-1), a pore diameter of 4.3 nm and a pore volume of 0.43 cm(3) g(-1). A slight decrease in surface area and pore volume was observed upon substitution with no distinct effect on pore diameter. In addition, all the MBG_Scs except 2.0% ZnO_Sc showed quite quick in vitro bioactive response. Hence, the present study is a positive addition to ongoing research into preparing bone tissue engineering scaffolds from bioceramics containing elements of therapeutic significance.

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.

Removal of cadmium ion by means of synthetic hydroxyapatite.

The reaction behaviour of synthetic hydroxyapatite [Ca10(PO4)6(OH)2] (HAP) toward cadmium ion was investigated for the Cd/Ca molar ratio in the range 1-0.005, by means of ions, pH measurements and XRD, SEM, IR techniques. The reaction behaviour between HAP and cadmium ion could be explained by a formation of an amorphous phase and/or a sorption mechanism.

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®.