Michele Alderighi

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.

Size effects in nanoindentation of hard and soft surfaces

Nanoindentation experiments carried out with atomic force microscopes (AFMs) open the way to understand size-related mechanical effects that are not present at the macro- or micro-scale. Several issues, currently the subject of a wide and open debate, must be carefully considered in order to measure quantities and retrieve trends genuinely associated with the material behaviour. The shape of the nanoindenter (the AFM tip) is crucial for a correct data analysis; we have recently developed a simple geometrical model to properly describe the tip effect in the nanoindentation process. Here, we demonstrate that this model is valid in indentation of both soft and hard, or relatively hard, materials carried out by two distinct, commercially available, AFM probes. Moreover, we implement the model with a data interpretation approach aimed at preventing underestimation of the tip penetration into the material. Experiments on soft polymeric materials (poly(methyl methacrylate) and polystyrene) and hard or relatively hard (Si, Au, Al) materials are reported. The results demonstrate that true hardness data can be attained also in shallow indentations and that the appearance of size effects strongly depends on data interpretation issues. In addition, we report on stiffness data measured on the considered materials during their nanoindentation.

Near-field optical microscopy of polymer-based films with dispersed terthiophene chromophores for polarizer applications

Linear dichroic properties of polyethylene films containing a dispersion of terthiophene-based chromophore molecules are investigated by using a scanning near-field optical microscope (SNOM). The polarization-modulation technique implemented in our SNOM provides quantitative information on the dichroic ratio of the samples with sub-wavelength space resolution. Optically active domains are identified and their morphology is analysed as a function of the film fabrication parameters, e.g., the drawing ratio and the kind of dispersed chromophore mixture. These investigations complement conventional polarimetry analysis by adding nanometre-scale information on the spatial distribution of the chromophore molecules and their mutual alignment with the host polymer chains.

Chemical-physical and in vivo evaluations of a self-assembling amphiphilic peptide as an injectable hydrogel scaffold for biomedical applications

The self-aggregation and gelation of an amphiphilic peptide (C17H35CONH–A4G3ERGD, peptide amphiphile) were studied by light scattering, viscometry, nuclear magnetic resonance diffusometry, and atomic force microscopy. The peptide amphiphile critical aggregation concentration was evaluated to be 16 and 60 µM by light scattering and viscometry, respectively. The observed difference was attributed to the larger sensitivity of the latter technique to the presence of long fibrils. The addition of one equivalent or more of divalent cations (Ca2+ and Mg2+) to peptide amphiphile formed dense incoherent hydrogels. Based on the atomic force microscopy and nanoindentation data, both the hydrogel morphology and stiffness were independent of the cation type and peptide amphiphile concentration. However, gel stiffness increased on increasing Ca2+/peptide amphiphile molar ratio while a parallel decrease in the apparent water diffusion rate was observed by nuclear magnetic resonance diffusometry. The dispersions of endothelial progenitor cells in the peptide amphiphile hydrogels were evaluated in vivo on a rat tissue hypoxia model. Significant capillary formation at the injection site was observed by tissue appearance and histological examination, which indicated endothelial progenitor cell/peptide amphiphile hydrogel-enhanced angiogenesis in ischemic tissue.

Dead Sea Minerals loaded polymeric nanoparticles

Therapeutic properties of Dead Sea Water (DSW) in the treatment of skin diseases such as atopic dermatitis, psoriasis and photo aging UV damaged skin have been well established. DSW is in fact rich in minerals such as calcium, magnesium, sodium, potassium, zinc and strontium which are known to exploit anti-inflammatory effects and to promote skin barrier recovery. In order to develop a Dead Sea Minerals (DSM) based drug delivery system for topical therapy of skin diseases, polymeric nanoparticles based on Poly (maleic anhydride-alt-butyl vinyl ether) 5% grafted with monomethoxy poly(ethyleneglycol) 2000 MW (PEG) and 95% grafted with 2-methoxyethanol (VAM41-PEG) loaded with DSM were prepared by means of a combined miniemulsion/solvent evaporation process. The resulting nanoparticles were characterized in terms of dimension, morphology, biocompatibility, salt content and release. Cytocompatible spherical nanoparticles possessing an average diameter of about 300 nm, a time controlled drug release profile and a high formulation yield were obtained.

AFM characterization of rabbit spermatozoa

Atomic force microscopy (AFM) has been applied for determining the topological and structural features of rabbit spermatozoa. Fresh ejaculated spermatozoa were adsorbed passively onto a silicon slide or by motility from suspension onto a poly(L‐lysine)‐coated glass coverslip and then imaged in air and in buffer saline, respectively. AFM images clearly highlighted many details of spermatozoa head, neck, and tail. Distinct features were observed in the plasmatic membrane of spermatozoa. In particular, head topography easily recognized the acrosome, equatorial segment, equatorial subsegment, and postacrosome regions. Moreover, AFM images revealed the presence of double belt of invaginations around the spermatozoa head, at the boundary between equatorial subsegment and postacrosome regions. All together, the collected AFM images clearly defined a detailed map of spermatozoa morphology while giving some hints on the internal structure. Microsc. Res. Tech., 2008.

Retinyl palmitate–loaded poly(lactide-co-glycolide) nanoparticles for the topical treatment of skin diseases

Nanoparticles were prepared with poly(lactide-co-glycolide), Pluronic F127, and phospholipids and loaded with retinyl palmitate. Morphology and physicochemical properties of these nanoparticles were determined by atomic force microscopy, light scattering, and zeta potential. The elasticity and deformability of the nanoparticles were correlated to Tg values measured by differential scanning calorimetry. The in vitro cytotoxicity and genotoxicity of the nanosystems were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, cell membrane asymmetry, and Ames tests with BALB/3T3 mouse embryo fibroblasts and HaCaT human keratinocytes cell lines. The reactive oxygen species levels and cytokine production in response to the exposure of cells to these nanoparticles were investigated, as well as the penetration in human skin culture.

All-optical pulsed writing in azobenzene copolymer films in the sub-millisecond regime

Block copolymers P(MA4-block-MMA)10 and P(MA4-block-MMA)20 and a random copolymer P(MA4-ran-MMA)10 were prepared from azobenzene methacrylate (MA4) and methyl methacrylate (MMA). The former monomer was introduced as both a photoresponsive and a mesogenic component, and the latter as a transparent non-mesogenic component. Thin films of the copolymers were used to investigate all-optical writing by linear-polarized illumination in the pulsed regime to modify the local birefringence at the microscopic scale. The optical properties of P(MA4-block-MMA)10 were compared with those of P(MA4-ran-MMA)10 having the same chemical composition (10 mol% MA4) but different distributions of the two components. It was found that in the block copolymers relatively intense pulses as short as 100 μs produced a remarkable local increase in the birefringence, stable in the time-scale (up to several days) explored in the experiment. In contrast, efficient and stable optical modifications could not be achieved in the random copolymer.

Hierarchical dual-sized film surface morphologies self-generated from fluorinated binary latex blends boost hydrophobicity and lipophobicity

Latex films with controlled dual-level nanorough surfaces were obtained by casting from binary blends of fluorinated copolymer particles with a nanostructured core-shell morphology, narrow size dispersity and large size ratios. For this purpose, particles with different size, a common unfluorinated acrylic core copolymer of the self-crosslinking trimethoxysilylpropyl methacrylate (TSPMA) and a hard shell copolymer of either 2,2,2-trifluoroethyl methacrylate (TFEMA) or 1H,1H,2H,2H-heptadecafluorodecyl methacrylate (FMA) were synthesized by multistage emulsion polymerization. The FMA-based particles showed patchy morphologies dictated by the type of β-cyclodextrin used as FMA phase carrier in their synthesis. Four series of binary blends of either TFEMA or FMA copolymer particles with large (3-4 diameters) size ratios were cast into latex films with controlled hydrophobicity and lipophobicity. AFM and electron microscopy results indicate that addition of the small particles disrupts the hexagonal compact packed 3D organization of the large particles, resulting in dual-level nanorough surfaces and high water contact angles (up to θ(w)=127° in the as cast films, and θ(w)=135° upon aging or thermal annealing causing surface restructuring and TSPMA sol-gel condensation) with respect to the parent single component films. The proposed approach provides a straightforward route for the fabrication of robust coatings and films with tunable lipophobic and highly hydrophobic surfaces.

Preparation and characterization of biodegradable amphiphilic polymers and nanoparticles with high protein-loading capacity

Multiblock copolymers containing carboxyl groups in the side-chains and at the chain ends were prepared from ABA triblock copolymers of ε-caprolactone, or lactide (as A block), and ethylene glycol (as B block). ABAn multiblock copolymers were prepared after chain-end functionalization and chain extension with pyromellitic dianhydride. A series of polymers were synthesized by varying the poly(ethylene glycol) and polyester molecular weight and the chirality of the lactide. Nuclear magnetic resonance analysis was used to confirm free carboxyl groups in the polymer backbone and at the chain ends. Thermal analysis indicated that the presence of pyromellitic dianhydride residues interfered not only with the formation of crystalline phases but also with the thermal degradation of chain-extended polymers. The biocompatibility of these amphiphilic polymers as evaluated with mouse embryo fibroblasts was acceptable. Both the parent ABA triblock copolymers and the carboxylated polymers were processed into nanoparticles. Depending on the polymer structure and reaction conditions, a narrow size nanoparticle distribution from ~10 to 250 nm was obtained. The nanoparticles were loaded with 60%–90% albumin and released 80%–90% of the albumin absorbed. Overall, this system was found to be well suited for the preparation of high-capacity injectable protein drug delivery.