Cristina Bartoli

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.

Poly(hydroxyalkanoates)-Based Polymeric Nanoparticles for Drug Delivery

Poly (hydroxyalkanoates) (PHAs) have recently attracted a great deal of academic and industrial interest for their biodegradability and biocompatibility making them suitable for environmental and biomedical applications. Poly(3-hydroxybutyrate-) (PHB-) and Poly(DL-lactide-co-glycolide) (PLGA-) based nanoparticles were prepared using the dialysis method as yet unreported for the preparation of nanoparticles based on PHB. Processing conditions were varied in order to evaluate their influence on morphology, drug encapsulation, and size of nanoparticles. The relevant results obtained give a theoretical understanding of the phenomenon occurring during colloidal formation. The adopted procedure allows for a relatively small diameter and homogeneity in size distribution of the PHB nanoparticles to be obtained compared to other methods like the one based on solvent evaporation which leads to particles on microscale. The biocompatibility of PHB and relative nanoparticles was investigated and both exhibited very good cytocompatibility.

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.

A new biocompatible nanoparticle delivery system for the release of fibrinolytic drugs

The preparation of novel biocompatible polymeric nanoconstructs suitable to load sensitive bioactive protein agents is reported. Nanoparticles were prepared as based on hybrid polymeric matrices consisting of synthetic bioerodible alternating copolymers of maleic anhydride and n-butylvinylether hemiesterified with 2-methoxyethanol and grafted with poly(ethylene glycol) segments and monoclonal antibody single chain fragment specific for fibrin clot. The prepared nanoparticles were loaded with proteolytic enzymes (trypsin and urokinase), encapsulating up to 2500UI of urokinase/mg of dried nanoparticles. The release of the enzyme from nanoparticles resulted time controlled and it was assessed that in case of administration of urokinase-loaded nanoparticles, the enzyme would preserve its thrombolytic properties more efficiently in respect to free drug administration. Moreover, the nanoparticles showed a good in vitro biocompatibility, suitable for biomedical applications. The stability (shelf life) of the prepared nanostructured dosage forms was evaluated. The drug-loaded nanoparticles resulted stable under stressed conditions (35 degrees C for 13 weeks) in a lyophilized form and preserved their morphological and functional characteristics when stored in suspension for 18 months at 4 degrees C.

Chitosan nanoparticles loaded with the antimicrobial peptide temporin B exert a long-term antibacterial activity in vitro against clinical isolates of Staphylococcus epidermidis

Nowadays, the alarming rise in multidrug-resistant microorganisms urgently demands for suitable alternatives to current antibiotics. In this regard, antimicrobial peptides (AMPs) have received growing interest due to their broad spectrum of activities, potent antimicrobial properties, unique mechanisms of action, and low tendency to induce resistance. However, their pharmaceutical development is hampered by potential toxicity, relatively low stability and manufacturing costs. In the present study, we tested the hypothesis that the encapsulation of the frog-skin derived AMP temporin B (TB) into chitosan nanoparticles (CS-NPs) could increase peptide’s antibacterial activity, while reducing its toxic potential. TB-loaded CS-NPs with good dimensional features were prepared, based on the ionotropic gelation between CS and sodium tripolyphosphate. The encapsulation efficiency of TB in the formulation was up to 75%. Release kinetic studies highlighted a linear release of the peptide from the nanocarrier, in the adopted experimental conditions. Interestingly, the encapsulation of TB in CS-NPs demonstrated to reduce significantly the peptide’s cytotoxicity against mammalian cells. Additionally, the nanocarrier evidenced a sustained antibacterial action against various strains of Staphylococcus epidermidis for at least 4 days, with up to 4-log reduction in the number of viable bacteria compared to plain CS-NPs at the end of the observational period. Of note, the antimicrobial evaluation tests demonstrated that while the intrinsic antimicrobial activity of CS ensured a “burst” effect, the gradual release of TB further reduced the viable bacterial count, preventing the regrowth of the residual cells and ensuring a long-lasting antibacterial effect. The developed nanocarrier is eligible for the administration of several AMPs of therapeutic interest with physical–chemical characteristics analog to those of TB.

Fibrous star poly(ε-caprolactone) melt-electrospun scaffolds for wound healing applications

Polymeric fibrous scaffolds based on the biocompatible and biodegradable three-arm-branched star poly(ε-caprolactone) (Mw = 189,000 g/mol) were prepared by a melt electrospinning technique. The possibility of processing polymers without the use of organic solvents is one of the main advantages over solution electrospinning. Scaffolds were biologically tested for their ability of supporting skin tissue regeneration. For this purpose, mouse embryo fibroblast (BALB/3T3 clone A31) and human keratinocyte (HaCaT) cell lines were selected as models, and seeded onto the polymeric supports both as single and co-culture. Cell viability, proliferation, and collagen production were assessed by WST-1 assay and Direct Red 80 dye, respectively. Cell morphology and colonization of the supports were evaluated by scanning electron microscopy and confocal laser scanning microscopy. Results highlighted that the star poly(ε-caprolactone) scaffolds were able to promote collagen production by fibroblasts. In co-culture studies, scaffolds supported adhesion, proliferation, and spatial organization of both cell lines. By virtue of the observed results, the developed polymeric scaffolds appeared suitable as biodegradable and biocompatible three-dimensional supports for skin tissue regeneration in wound healing dressing.

Bioactive polymeric materials for targeted administration of active agents: synthesis and evaluation.

Bioerodible polymers displaying both stealth and targeting properties for the preparation of nanosystems for targeted and controlled delivery of fibrinolytic drugs to the thrombus were prepared by straightforward synthetic routes and characterized. Poly[(maleic anhydride)-alt-(butylvinyl ether)]s were synthesized in the presence of dodecyl mercaptan as chain transfer agent allowing for the preparation of copolymers with tunable molecular weight. 2-Methoxyethanol hemiesters containing antiopsonizing molecules of poly(ethylene glycol) were prepared and further biofunctionalized with a Fab fragment by a two-step reaction. In vitro biocompatibility investigation of the prepared materials supported their suitability for biomedical applications.

Microstructured chitosan/poly(γ-glutamic acid) polyelectrolyte complex hydrogels by computer-aided wet-spinning for biomedical three-dimensional scaffolds

The application of additive manufacturing principles to hydrogel processing represents a powerful route to develop porous three-dimensional constructs with a variety of potential biomedical applications, such as scaffolds for tissue engineering and three-dimensional in vitro tissue models. The aim of this study was to develop novel porous hydrogels based on a microstructured polyelectrolyte complex between chitosan and poly(γ-glutamic acid) by applying a computer-aided wet-spinning technique. The developed fabrication process was used to build up three-dimensional porous hydrogels by collecting microstructured layers made of chitosan/poly(γ-glutamic acid) on top of the other. Microstructured polyelectrolyte complex hydrogels were characterized and compared to chitosan/poly(γ-glutamic acid) porous hydrogels with similar composition prepared by conventional freeze-drying technique. Fourier transform infrared analysis confirmed the formation of an electrostatic interaction between the two processed polymers in all the developed chitosan/poly(γ-glutamic acid) hydrogels. The composition of the porous constructs as well as the employed processing techniques had a significant influence on the resulting swelling, thermal, and mechanical properties. In particular, the combination of the ionic interaction between chitosan/poly(γ-glutamic acid) and the defined internal microarchitecture of microstructured polyelectrolyte complex hydrogels provided a significant improvement of the compressive mechanical properties. Preliminary in vitro biological investigations revealed that microstructured polyelectrolyte complex hydrogels were suitable for the adhesion and proliferation of Balb/3T3 clone A31 mouse embryo fibroblasts. The encouraging results in terms of cytocompatibility and stability of the microstructure in aqueous solutions due to the ionic crosslinking suggest the investigation of the developed microstructured polyelectrolyte complex hydrogels as suitable scaffolds for three-dimensional cells’ culture.

Mg- and/or Sr-doped tricalcium phosphate/bioactive glass composites: Synthesis, microstructure and biological responsiveness

Presently, there is an increasing interest towards the composites of calcium phosphates, especially β-tricalcium phosphate (TCP), and bioactive glasses. In the present contribution, the recently developed BG_Ca/Mix glass has been used because its low tendency to crystallize allows to sinter the composites at relatively low temperature (i.e. 850°C), thus minimizing the glass devitrification and the interaction with TCP. A further improvement is the introduction of lab-produced TCP powders doped with specific ions instead of non-doped commercial powders, since the biological properties of materials for bone replacement can be modulated by doping them with certain metallic ions, such as Mg and Sr. Therefore, novel binary composites have been produced by sintering the BG_Ca/Mix glass with the addition of pure, Mg-substituted, Sr-substituted or Mg/Sr bisubstituted TCP powders. After an accurate characterization of the starting TCP powders and of the obtained samples, the composites have been used as three-dimensional supports for the culture of mouse calvaria-derived pre-osteoblastic cells. The samples supported cell adhesion and proliferation and induced promising mechanisms of differentiation towards an osteoblastic phenotype. In particular, the Mg/Sr bi-doped samples seemed to better promote the differentiation process thus suggesting a combined stimulatory effect of Mg(2+) and Sr(2+) ions.