Multiscale static and dynamic mechanical study of the Turritella terebra and Turritellinella tricarinata seashells.

Marine shells are designed by nature to ensure mechanical protection from predators and shelter for molluscs living inside them. A large amount of work has been done to study the multiscale mechanical properties of their complex microstructure and to draw inspiration for the design of impact-resistant biomimetic materials. Less is known regarding the dynamic behaviour related to their structure at multiple scales. Here, we present a combined experimental and numerical study of the shells of two different species of gastropod sea snail belonging to the Turritellidae family, featuring a peculiar helicoconic shape with hierarchical spiral elements. The proposed procedure involves the use of micro-computed tomography scans for the accurate determination of geometry, atomic force microscopy and nanoindentation to evaluate local mechanical properties, surface morphology and heterogeneity, as well as resonant ultrasound spectroscopy coupled with finite element analysis simulations to determine global modal behaviour. Results indicate that the specific features of the considered shells, in particular their helicoconic and hierarchical structure, can also be linked to their vibration attenuation behaviour. Moreover, the proposed investigation method can be extended to the study of other natural systems, to determine their structure-related dynamic properties, ultimately aiding the design of bioinspired metamaterials and of structures with advanced vibration control.

Pressure-activated microsyringe (PAM) fabrication of bioactive glass-poly(lactic-co-glycolic acid) composite scaffolds for bone tissue regeneration.

The aim of this work was the fabrication and characterization of bioactive glass-poly(lactic-co-glycolic acid) (PLGA) composite scaffolds mimicking the topological features of cancellous bone. Porous multilayer PLGA-CEL2 composite scaffolds were innovatively produced by a pressure-activated microsyringe (PAM) method, a CAD/CAM processing technique originally developed at the University of Pisa. In order to select the optimal formulations to be extruded by PAM, CEL2-PLGA composite films (CEL2 is an experimental bioactive SiO2 -P2 O5 -CaO-MgO-Na2 O-K2 O glass developed at Politecnico di Torino) were produced and mechanically tested. The elastic modulus of the films increased from 30 to > 400 MPa, increasing the CEL2 amount (10-50 wt%) in the composite. The mixture containing 20 wt% CEL2 was used to fabricate 2D and 3D bone-like scaffolds composed by layers with different topologies (square, hexagonal and octagonal pores). It was observed that the increase of complexity of 2D topological structures led to an increment of the elastic modulus from 3 to 9 MPa in the composite porous monolayer. The elastic modulus of 3D multilayer scaffolds was intermediate (about 6.5 MPa) between the values of the monolayers with square and octagonal pores (corresponding to the lowest and highest complexity, respectively). MG63 osteoblast-like cells and periosteal-derived precursor cells (PDPCs) were used to assess the biocompatibility of the 3D bone-like scaffolds. A significant increase in cell proliferation between 48 h and 7 days of culture was observed for both cell phenotypes. Moreover, qRT-PCR analysis evidenced an induction of early genes of osteogenesis in PDPCs. Copyright © 2015 John Wiley & Sons, Ltd.

Phosphate glass fibre scaffolds: Tailoring of the properties and enhancement of the bioactivity through mesoporous glass particles.

Novel bone glass fibre scaffolds were developed by thermally bonding phosphate glass fibres belonging to the P2O5-CaO-Na2O-SiO2-MgO-K2O-TiO2 system (TiPS2.5 glass). Scaffolds with fibres of 85 or 110µm diameter were fabricated, showing compressive strength in the range of 2-3.5MPa, comparable to that of the trabecular bone. The effect of different thermal treatments and fibre diameters and length on the final scaffold structure was investigated by means of micro-CT analysis. The change of the sintering time from 30 to 60min led to a decrease in the scaffold overall porosity from 58 to 21vol.% for the 85µm fibre scaffold and from 50 to 40vol.% when increasing the sintering temperature from 490 to 500°C for the 110µm fibre scaffold. The 85µm fibres resulted in an increase of the scaffold overall porosity, increased pore size and lower trabecular thickness; the use of different fibre diameters allowed the fabrication of a scaffold showing a porosity gradient. In order to impart bioactive properties to the scaffold, for the first time in the literature the introduction in these fibre scaffolds of a bioactive phase, a melt-derived bioactive glass (CEL2) powder or spray-dried mesoporous bioactive glass particles (SD-MBG) was investigated. The scaffold bioactivity was assessed through soaking in simulated body fluid. CEL2/glass fibre scaffold did not show promising results due to particle detachment from the fibres during soaking in simulated body fluid. Instead the use of mesoporous bioactive powders showed to be an effective way to impart bioactivity to the scaffold and could be further exploited in the future through the ability of mesoporous particles to act as systems for the controlled release of drugs.

Bioactivity and Mechanical Stability of 45S5 Bioactive Glass Scaffolds Based on Natural Marine Sponges.

Bioactive glass (BG) based scaffolds (45S5 BG composition) were developed by the replica technique using natural marine sponges as sacrificial templates. The resulting scaffolds were characterized by superior mechanical properties (compression strength up to 4 MPa) compared to conventional BG scaffolds prepared using polyurethane (PU) packaging foam as a template. This result was ascribed to a reduction of the total scaffold porosity without affecting the pore interconnectivity (>99%). It was demonstrated that the reduction of total porosity did not affect the bioactivity of the BG-based scaffolds, tested by immersion of scaffolds in simulated body fluid (SBF). After 1 day of immersion in SBF, a homogeneous CaP deposit on the surface of the scaffolds was formed, which evolved over time into carbonate hydroxyapatite (HCA). Moreover, the enhanced mechanical properties of these scaffolds were constant over time in SBF; after an initial reduction of the maximum compressive strength upon 7 days of immersion in SBF (to 1.2 ± 0.2 MPa), the strength values remained almost constant and higher than those of BG-based scaffolds prepared using PU foam (<0.05 MPa). Preliminary cell culture tests with Saos-2 osteoblast cell line, namely direct and indirect tests, demonstrated that no toxic residues remained from the natural marine sponge templates and that cells were able to proliferate on the scaffold surfaces.

Novel systems for tailored neurotrophic factor release based on hydrogel and resorbable glass hollow fibers.

A novel system for the release of neurotrophic factor into a nerve guidance channel (NGC) based on resorbable phosphate glass hollow fibers (50P2O5-30CaO-9Na2O-3SiO2-3MgO-2.5K2O-2.5TiO2 mol%) in combination with a genipin-crosslinked agar/gelatin hydrogel (A/G_GP) is proposed. No negative effect on the growth of neonatal olfactory bulb ensheathing cell line (NOBEC) as well as on the expression of pro- and anti-apoptotic proteins was measured in vitro in the presence of fiber dissolution products in the culture medium. For the release studies, fluorescein isothiocyanate-dextran (FD-20), taken as growth factor model molecule, was solubilized in different media and introduced into the fiber lumen exploiting the capillary action. The fibers were filled with i) FD-20/phosphate buffered saline (PBS) solution, ii) FD-20/hydrogel solution before gelation and iii) hydrogel before gelation, subsequently lyophilized and then filled with the FD-20/PBS solution. The different strategies used for the loading of the FD-20 into the fibers resulted in different release kinetics. A slower release was observed with the use of A/G_GP hydrogel. At last, poly(ε-caprolactone) (PCL) nerve guides containing the hollow fibers and the hydrogel have been fabricated.

Phosphate glass fibres and their role in neuronal polarization and axonal growth direction.

Phosphate glass fibres with composition 50P(2)O(5)-30CaO-9Na(2)O-3SiO(2)-3MgO-(5-x)K(2)O-xTiO(2)mol.% (x=0, 2.5, 5, respectively coded as TiPS(0), TiPS(2.5) and TiPS(5)) were drawn following the preform drawing approach. A 20-day solubility test in bi-distilled water was carried out on glass fibres with different compositions and diameters ranging between 25 and 82 µm. The results show that the glass composition, the initial fibre diameter and the thermal treatment are the main factors influencing the dissolution kinetics and that the fibres maintain their structural integrity and composition during dissolution. Biological tests were carried out on aligned TiPS(2.5) glass fibres using Neonatal Olfactory Bulb Ensheathing Cell Line (NOBEC) and Dorsal Root Ganglia (DRG) neurons. The fibres showed to be permissive substrates for cell adhesion and proliferation. The aligned configuration of the fibres seemed to provide a directional cue for growing axons of DRG neurons, which showed to sprout and grow long neurites along the fibre axis direction. These promising findings encourages further studies to evaluate the potential use of resorbable glass fibres (e.g.in combination with a nerve guidance tube) for the enhancement of the peripheral nerve healing with the role of supporting and guiding the cells involved in the nerve regeneration.

Effects of TiO2-containing phosphate glasses on solubility and in vitro biocompatibility.

Phosphate-based glasses with different amounts of titanium dioxide (TiO(2)), having the following molar composition 50P(2)O(5)-30CaO-9Na(2)O-3SiO(2)-3MgO-(5-x)K(2)O-xTiO(2), (where x = 0, 2.5, 5 mol %), were synthesised and characterized in terms of solubility (according to ISO 10993-14), and in vitro biocompatibility using human MG-63 osteoblast cells. Dissolution tests were carried out in Tris-HCl (pH 7.4) to simulate the physiological pH and in citric acid (pH 3.0) to simulate an acidic environment. The weight loss decreased with increasing TiO(2) content, a process further enhanced in acidic medium. TiO(2) reduced the pH changes usually caused by the dissolution products released. Cellular tests showed that all the glasses studied (0-5 mol % TiO(2)) and TiCl(4), used to investigate the biocompatibility of titanium ions, did not produce cytotoxic effects on human MG-63 osteoblasts for up to 5 days in culture. On the basis of these results, we suggest that TiO(2)-containing phosphate glasses could be promising substrates for bone tissue engineering applications.

Response of human bone marrow stromal cells to a resorbable P(2)O(5)-SiO(2)-CaO-MgO-Na(2)O-K(2)O phosphate glass ceramic for tissue engineering applications.

This work focuses on the synthesis and characterization of a novel bioresorbable glass ceramic phosphate-based material (GC-ICEL). More specifically, its solubility in different aqueous media (water, Tris-HCl and acellular simulated body fluid) and the response of human stromal cells cultured on it were investigated. X-ray diffraction analysis showed the presence of two crystalline phases identified as Na(2)Mg(PO(4))(3) and Ca(2)P(2)O(7) and dissolution tests highlighted a preferential dissolution of the Na(2)Mg(PO(4))(3) phase and of the residual amorphous phase in all the chosen media. Soaking tests in simulated body fluid showed precipitation of a hydroxyapatite layer, demonstrating the bioactivity of GC-ICEL, which is partially due to the reported bioactivity of Ca(2)P(2)O(7). The effect of GC-ICEL on adhesion, proliferation and osteoblastic gene expression of human bone marrow-derived stromal cells was also studied. Combining molecular and biochemical analyses, it was found that bone marrow cell differentiation was stimulated over proliferation on GC-ICEL. Moreover, the expression of bone-related genes in cells cultured on GC-ICEL confirmed the bioactivity of this phosphate-based glass ceramic, which might have a stimulatory effect on osteogenesis.

Surface silver-doping of biocompatible glasses to induce antibacterial properties. Part II: Plasma sprayed glass-coatings.

A 57% SiO(2), 3% Al(2)O(3), 34% CaO and 6% Na(2)O glass (SCNA) has been produced in form of powders and deposited by plasma spray on titanium alloy and stainless steel substrates. The obtained coatings have been subjected to a patented ion-exchange treatment to introduce silver ions in the surface inducing an antibacterial behavior. Silver surface-enriched samples have been characterized by means of X-ray diffraction, SEM observation, EDS analysis, in vitro bioactivity tests, leaching tests by GFAAS (graphite furnace atomic adsorption spectroscopy) analyses, cells adhesion and proliferation, and antibacterial tests using Staphylococcus Aureus strain. In vitro tests results showed that the modified samples acquired an antimicrobial action against tested bacteria maintaining unaffected the biocompatibility of the glass. Furthermore the ion-exchange treatment can be successfully applied to glass-coated samples without affecting the properties of the coatings; the simplicity and reproducibility of the method make it suitable for glass or glass-ceramic coatings of different composition in order to produce coated devices for bone healing and/or prostheses, able to reduce bacterial colonization and infections risks.

Surface silver-doping of biocompatible glass to induce antibacterial properties. Part I: Massive glass.

A glass belonging to the system SiO(2)-Al(2)O(3)-CaO-Na(2)O has been subjected to a patented ion-exchange treatment to induce surface antibacterial activity by doping with silver ions. Doped samples have been characterized by means of X-Ray diffraction (XRD), scanning electron microscopy (SEM) observation, energy dispersion spectrometry (EDS) analysis, in vitro bioactivity test, Ag(+) leaching test by graphite furnace atomic absorption spectroscopy (GFAAS) analyses, cytotoxicity tests by fibroblasts adhesion and proliferation, adsorption of IgA and IgG on to the material to evaluate its inflammatory property and antibacterial tests (cultures with Staphylococcus aureus and Escherichia coli). In vitro tests results demonstrated that the modified glass maintains the same biocompatibility of the untreated one and, moreover, it acquires an antimicrobial action against tested bacteria. This method can be selected to realize glass or glass-ceramic bone substitutes as well as coatings on bio-inert devices, providing safety against bacterial colonization thus reducing the risks of infections nearby the implant site. The present work is the carrying on of a previous research activity, concerning the application of an ion-exchange treatment on glasses belonging to the ternary system SiO(2)-CaO-Na(2)O. On the basis of previous results the glass composition was refined and the ion-exchange process was adapted to it, in order to tune the final material properties. The addition of Al(2)O(3) to the original glass system and the optimization of the ion-exchange conditions allowed a better control of the treatment, leading to an antibacterial material, without affecting both bioactivity and biocompatibility.

Surface functionalization of bioactive glasses.

Different cleaning and silanization methods have been applied to bioactive glasses with the aim of covalently bonding bone morphogenetic proteins (BMP-2) to the surface. Several glasses, with different bioactivity index, were cleaned with acidic, basic, or neutral aqueous media to investigate the role of pH in the formation of silanols on glass surfaces of different reactivity. The cleaned glasses were then functionalized using 3-aminopropyl-triethoxysilane (APTS). After the optimization of the silanization procedure, proteins of different complexity were immobilized on the functionalized glasses. To optimize the protein immobilization, a model protein (carnosine) was first used, and the procedure was then used to bind human BMP-2. The glass surfaces were characterized during each step of the treatment by water contact angles and X-ray photoelectron analyses. The APTS functionalization was then used to immobilize bone morphogenetic protein on the bioactive glasses. This result suggested that such a treatment could be successfully used as an efficient alternative to systemic administration of transforming growth factors for the development of local delivery vehicle implants.

Early stage reactivity and in vitro behavior of silica-based bioactive glasses and glass-ceramics.

The surface reactivity of different sets of glasses and glass-ceramics belonging to the SiO(2)-P(2)O(5)-CaO-MgO-K(2)O-Na(2)O system have been investigated. The attention was focused on the role of their composition on the bioactivity kinetics, in terms of pH modifications, silica-gel formation and its evolution toward hydroxycarbonatoapatite, after different times of soaking in simulated body fluid. Glasses and glass ceramics have been characterized by thermal analysis, SEM-EDS observations and phase analysis (XRD). XPS measurements have been carried out on the most representative set of sample in order to evaluate the evolution of the surface species during the growth of silica-gel and hydroxycarbonatoapatite. The response of murine fibroblast 3T3 to the material before and after a conditioning pre-treatment (immersion in SBF) has been investigated on the same set of samples in order to point out the role of the bioactivity mechanism on cell viability. The main differences among the various glasses have been related to the modifier oxides ratio and to the MgO content, which seems to have an influence on the glass stability, both in terms of thermal properties and surface reactivity. The surface characterization and in vitro tests revealed few variations in the reactivity of the different glasses and glass-ceramics in their pristine form. On the contrary, the different surface properties before and after the pre-treatment in SBF seem to play a role on the biocompatibility of both glass and glass-ceramics, due to the different ion release and hydrophilicity of the surfaces, affecting both cell viability and protein adsorption.

SBA-15 ordered mesoporous silica inside a bioactive glass-ceramic scaffold for local drug delivery.

The paper reports the synthesis of an ordered silica mesostructure of the SBA-15 type inside a macroporous bioactive glass-ceramic scaffold of the type SiO(2)-CaO-K(2)O, to combine the bioactivity of the latter with the release properties of the former, in view of local drug delivery from implants designed for tissue engineering. The standard procedure for SBA-15 synthesis has been modified to minimize the damage to the scaffold caused by the acidic synthesis medium. The composite system has been characterized by means of Scanning Electron Microscopy (coupled with EDS analysis), Small Angle X-Ray Diffraction, Thermogravimetry analysis and Infrared Spectroscopy: the formation of a well ordered hexagonal mesostructure was confirmed. Ibuprofen has been chosen as model drug. The uploading properties have been investigated of the scaffold-mesoporous silica composite as compared with the scaffold as such, and a five-fold increase in the adsorbing properties toward ibuprofen was found, due to the presence of the ordered mesoporous silica. The ibuprofen release to a SBF solution in vitro is complete in 1 day. Retention of bioactivity from the glass-ceramic scaffold after the silica mesostructure incorporation has been observed.

Development of glass-ceramic scaffolds for bone tissue engineering: characterisation, proliferation of human osteoblasts and nodule formation.

Glass-ceramic macroporous scaffolds for tissue engineering have been developed using a polyurethane sponge template and bioactive glass powders. The starting glass (CEL2) belongs to the system SiO(2)-P(2)O(5)-CaO-MgO-Na(2)O-K(2)O and has been synthesised by a conventional melting-quenching route. A slurry of CEL2 powder, polyvinyl alcohol and water has been prepared in order to coat, by impregnation, the polymeric template. An optimised thermal treatment was then use to remove the sponge and to sinter the glass powders, leading to a glass-ceramic replica of the template. Morphological observations, image analyses, mechanical tests and in vitro tests showed that the obtained devices are good candidates as scaffolds for bone-tissue engineering, in terms of pore-size distribution, pore interconnection, surface roughness, and both bioactivity and biocompatibility. In particular, a human osteoblast cell line (MG-63) seeded onto the scaffold after a standardised preconditioning route in simulated body fluid showed a high degree of cell proliferation and a good ability to produce calcium nodules. The obtained results were enhanced by the addition of bone morphogenetic proteins after cell seeding.

Microstructural and in vitro characterization of SiO2-Na2O-CaO-MgO glass-ceramic bioactive scaffolds for bone substitutes.

In the present research work, the preparation and characterization of bioactive glass-ceramic scaffolds for bone substitutes are described. The scaffolds were prepared by starch consolidation of bioactive glass powders belonging to the SiO2-Na2O-CaO-MgO system using three different organic starches (corn, potatoes and rice) as reported in a previous screening process. The scaffolds, characterized by scanning electron microscopy, showed a porous structure with highly interconnected pores. The pores sizes assessed by mercury intrusion porosimetry put in evidence the presence of pores of 50-100 microm. The structure of the scaffolds was investigated by X-ray diffraction and revealed the glass-ceramic nature of the obtained material. The mechanical properties of the scaffolds were evaluated by means of compressive tests on cubic samples and the obtained results demonstrated their good mechanical strength. The in vitro bioactivity of the scaffolds was tested by soaking them in a simulated body fluid (SBF) and by subsequently characterizing the soaked surfaces by SEM, EDS and X-ray diffraction. Good in vitro bioactivity was found for the starting glass and for the obtained scaffolds. Moreover, the scaffold bioresorption, tested by measuring the samples weight loss in SBF at different periods of time, showed a partial resorption of the scaffolds. Cell culture testing of the three different scaffolds indicated no differences in cell number and in alkaline phosphatase activity; the morphology of the osteoblasts showed good spreading, comparable to bulk material which was used as the control.

SiO2-CaO-K2O coatings on alumina and Ti6Al4V substrates for biomedical applications.

Alumina and Ti6Al4V alloys are widely used for orthopedics and dental applications due to their good mechanical properties and biocompatibility. Unfortunately they can not provide a satisfactory osteointegration when implanted. In fact, both alumina and Ti6Al4V are not bioactive and thus they can only guarantee a morphological fixation with the surrounding tissues without a suitable chemical anchorage. Aiming to impart bioactive properties to these materials a coating can be proposed. At this purpose, a bioactive glass belonging to the SiO2-CaO-K2O system was selected and prepared. This glass, named SCK, possess a thermal expansion coefficient matching with the alumina (8.5x 10(- 6)/ degrees C) and Ti6Al4V (9 x 10(- 6)/ degrees C) ones and thus is a good candidate to produce coatings on both of them. Simple and low-cost enameling and glazing techniques were used to realize the coatings. Structural, morphological and compositional characterizations of the coatings were carried out by means of X-ray diffraction, optical and scanning microscopy and compositional analyses. The in vitro properties of the coatings were investigated by soaking them in a simulated body fluid (SBF) in order to study the precipitation, on their surfaces, of a biologically active layer of hydroxylapatite (HAp).

Macroporous glass-ceramic materials with bioactive properties.

In the present research work, glass powders and three different organic starches were used to realize macroporous glass-ceramic scaffolds for bone substitutions. For this purpose, bioactive glass powders belonging to the system SiO2-CaO-Na2O-MgO were mixed in a liquid medium with the desired amount of the selected organic phase. Afterwards, by progressively raising the temperature, the water uptake of starches occurred and led to the gelling of the whole system. The resultant gel underwent two thermal treatments in order to eliminate the organic phase and to allow the sintering of the glassy phase. In this way, macroporous glass-ceramic scaffolds were successfully prepared. The samples were characterized by means of optical and scanning electron microscopy with compositional analysis. The volume and mean size of the obtained porosity were investigated by means of mercury intrusion porosimetry, whereas its morphology was assessed by means of microscopic observations. The structure of the original and the resultant materials were investigated by X-ray diffraction. In order to study the reactivity of the scaffolds towards physiological media, the samples were soaked in a simulated body fluid for various times. On their soaked surfaces, scanning electron microscopy and compositional analysis were carried out in order to assess their bioactivity.