o Dinucci

A simple approach to covalent functionalization of boron nitride nanotubes

A novel and simple method for the preparation of chemically functionalized boron nitride nanotubes (BNNTs) is presented. Thanks to a strong oxidation followed by the silanization of the surface through 3-aminopropyl-triethoxysilane (APTES), BNNTs exposing amino groups on their surface were successfully obtained. The efficacy of the procedure was assessed with EDS and XPS analyses, which demonstrated a successful functionalization of ~15% boron sites. This approach opens interesting perspectives for further modification of BNNTs with several kinds of molecules. Since, in particular, biomedical applications are envisaged, we also demonstrated in vitro biocompatibility and cellular up-take of the functionalized BNNTs.

Investigation of interactions between poly-l-lysine-coated boron nitride nanotubes and C2C12 cells: up-take, cytocompatibility, and differentiation

Boron nitride nanotubes (BNNTs) have generated considerable interest within the scientific community by virtue of their unique physical properties, which can be exploited in the biomedical field. In the present in vitro study, we investigated the interactions of poly-l-lysine-coated BNNTs with C2C12 cells, as a model of muscle cells, in terms of cytocompatibility and BNNT internalization. The latter was performed using both confocal and transmission electron microscopy. Finally, we investigated myoblast differentiation in the presence of BNNTs, evaluating the protein synthesis of differentiating cells, myotube formation, and expression of some constitutive myoblastic markers, such as MyoD and Cx43, by reverse transcription – polymerase chain reaction and Western blot analysis. We demonstrated that BNNTs are highly internalized by C2C12 cells, with neither adversely affecting C2C12 myoblast viability nor significantly interfering with myotube formation.

Polycaprolactone Scaffolds Fabricated via Bioextrusion for Tissue Engineering Applications

The most promising approach in Tissue Engineering involves the seeding of porous, biocompatible/biodegradable scaffolds, with donor cells to promote tissue regeneration. Additive biomanufacturing processes are increasingly recognized as ideal techniques to produce 3D structures with optimal pore size and spatial distribution, providing an adequate mechanical support for tissue regeneration while shaping in-growing tissues. This paper presents a novel extrusion-based system to produce 3D scaffolds with controlled internal/external geometry for TE applications.The BioExtruder is a low-cost system that uses a proper fabrication code based on the ISO programming language enabling the fabrication of multimaterial scaffolds. Poly(ε-caprolactone) was the material chosen to produce porous scaffolds, made by layers of directionally aligned microfilaments. Chemical, morphological, and in vitro biological evaluation performed on the polymeric constructs revealed a high potential of the BioExtruder to produce 3D scaffolds with regular and reproducible macropore architecture, without inducing relevant chemical and biocompatibility alterations of the material.

Porous scaffolds of polycaprolactone reinforced with in situ generated hydroxyapatite for bone tissue engineering.

Polycaprolactone/hydroxyapatite (PCL/HA) composites were prepared by in situ generation of HA in the polymer solution starting from the precursors calcium nitrate tetrahydrate and ammonium dihydrogen phosphate via sol–gel process. Highly interconnected porosity was achieved by means of the salt-leaching technique using a mixture of sodium chloride and sodium bicarbonate as porogens. Structure and morphology of the PCL/HA composites were investigated by scanning electron microscopy, and mechanical properties were determined by means of tensile and compression tests. The possibility to employ the developed composites as scaffolds for bone tissue regeneration was assessed by cytotoxicity test of the PCL/HA composites extracts and cell adhesion and proliferation in vitro studies.

Novel electrospun polyurethane/gelatin composite meshes for vascular grafts

Novel polymeric micro-nanostructure meshes as blood vessels substitute have been developed and investigated as a potential solution to the lack of functional synthetic small diameter vascular prosthesis. A commercial elastomeric polyurethane (Tecoflex® EG-80A) and a natural biopolymer (gelatin) were successfully co-electrospun from different spinnerets on a rotating mandrel to obtain composite meshes benefiting from the mechanical characteristics of the polyurethane and the natural biopolymer cytocompatibility. Morphological analysis showed a uniform integration of micrometric (Tecoflex®) and nanometric (gelatin) fibers. Exposure of the composite meshes to vapors of aqueous glutaraldehyde solution was carried out, to stabilize the gelatin fibers in an aqueous environment. Uniaxial tensile testing in wet conditions demonstrated that the analyzed Tecoflex®–Gelatin specimens possessed higher extensibility and lower elastic modulus than conventional synthetic grafts, providing a closer matching to native vessels. Biological evaluation highlighted that, as compared with meshes spun from Tecoflex® alone, the electrospun composite constructs enhanced endothelial cells adhesion and proliferation, both in terms of cell number and morphology. Results suggest that composite Tecoflex®–Gelatin meshes could be promising alternatives to conventional vascular grafts, deserving of further studies on both their mechanical behaviour and smooth muscle cell compatibility.

Involvement of cytochrome P450 2E1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease.

Elucidation of the biochemical steps leading to the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced degeneration of the nigrostriatal dopamine (DA) pathway has provided new clues to the pathophysiology of Parkinsons disease. In line with the enhancement of MPTP toxicity by diethyldithiocarbamate (DDC), here we demonstrate how other cytochrome P450 (CYP) 2E1 inhibitors, such as diallyl sulphide (DAS) and phenylethylisothiocyanate (PIC), also potentiate the selective DA neurone degeneration in C57/bl mice. In addition, we show that CYP 2E1 is present in the brain and in the basal ganglia of this mouse strain, as measured by RT–PCR, western blot analysis and immunohistochemistry. A kinetic analysis of MPTP and its metabolites, by means of the microdialysis technique in the striatum, indicates that no detoxification metabolic pathway is affected by any of these inhibitors. This does not rule out, however, that an undetected detoxification pathway involving CYP 2E1 is operating. In order to provide direct evidence for this isozyme involvement, CYP 2E1 knockout mice were challenged with MPTP or the combined treatment. Here we show that these transgenic mice have a low sensitivity to MPTP alone, similar to their wild‐type counterparts, suggesting that it is likely that transgenic mice compensate for the missing enzyme. However, DDC pretreatment completely fails to enhance MPTP toxicity in CYP 2E1 knockout mice, whereas this enhancement is regularly present in wild‐type animals. This study indicates that the occurrence of CYP 2E1 in C57/bl mouse brain is relevant to MPTP toxicity, and suggests that this isozyme may have a detoxificant role related to the efflux transporter of the toxin.

Development of 3D wet-spun polymeric scaffolds loaded with antimicrobial agents for bone engineering

Three-dimensional wet-spun microfibrous meshes of a star poly(∈-caprolactone) were developed as potential scaffolds endowed with antimicrobial activity. The in vitro release kinetics of the meshes, under physiological conditions, was initially fast and then a sustained release for more than one month was observed. Cell cultures of a murine pre-osteoblast cell line showed good cell viability and adhesion on the wet-spun star poly(∈-caprolactone) fiber scaffolds. These promising results indicate a potential application of the developed meshes as engineered bone scaffolds with antimicrobial activity.

Fibrin acts as biomimetic niche inducing both differentiation and stem cell marker expression of early human endothelial progenitor cells

Objectives:  Transplantation of endothelial progenitor cells (EPCs) is a promising approach for revascularization of tissue. We have used a natural and biocompatible biopolymer, fibrin, to induce cell population growth, differentiation and functional activity of EPCs.

Additive manufacturing of star poly(ε-caprolactone) wet-spun scaffolds for bone tissue engineering applications

Three-dimensional fibrous scaffolds made of a three-arm star poly(ε-caprolactone) were developed by employing a novel computer-aided wet-spinning apparatus to precisely control the deposition pattern of an extruded polymeric solution as a filament into a coagulation bath. Star poly(ε-caprolactone)/hydroxyapatite composite scaffolds composed of fibres with a porous morphology both in the outer surface and in the cross section were successfully produced with a layer-by-layer approach achieving good reproducibility of the internal architecture and external shape. Changes in processing parameters were used to fabricate scaffolds with different architectural parameters in terms of average pore size in the xy-axes (from 190 to 297 µm) and in the z-axis (from 54 to 126 µm) and porosity (in the range of 20%–60%). Based on the mechanical characterization, processing variations and hydroxyapatite loading have an influence on scaffold compression properties. Cell cultures, using a murine pre-osteoblast cell line, had good cell responses in terms of proliferation and osteoblastic differentiation. Thus, this technique appears to be an effective method for producing customized polymeric scaffolds for bone tissue engineering applications.

Dual-Scale Polymeric Constructs as Scaffolds for Tissue Engineering

This research activity was aimed at the development of dual-scale scaffolds consisting of three-dimensional constructs of aligned poly(ε-caprolactone) (PCL) microfilaments and electrospun poly(lactic-co-glycolic acid) (PLGA) fibers. PCL constructs composed by layers of parallel microsized filaments (0/90° lay-down pattern), with a diameter of around 365 μm and interfilament distance of around 191 μm, were produced using a melt extrusion-based additive manufacturing technique. PLGA electrospun fibers with a diameter of around 1 μm were collected on top of the PCL constructs with different thicknesses, showing a certain degree of alignment. Cell culture experiments employing the MC3T3 murine preosteoblast cell line showed good cell viability and adhesion on the dual-scale scaffolds. In particular, the influence of electrospun fibers on cell morphology and behavior was evident, as well as in creating a structural bridging for cell colonization in the interfilament gap.