Lorenzo Ferraro

Wear effects on microscopic morphology and hyaluronan uptake in siloxane-hydrogel contact lenses

The purpose of this study was a comparison between new and worn siloxane-hydrogel contact lenses in terms of microscopic structure, surface morphology, and loading of hyaluronan. The analyses were performed by scanning electron microscopy, with the support of the freeze-drying technique, and by fluorescence confocal microscopy. Along the depth profile of new lenses, a thin porous top layer was observed, which corresponds to the region of hyaluronan penetration inside well-defined channels. The time evolution was followed from one day to two weeks of daily wear, when a completely different scenario was found. Clear experimental evidence of a buggy surface was observed with several crests and regions of swelling, which could be filled by the hyaluronan solution. The modifications are attributed to the progressive relaxation of the structure of the polymeric network.

Magnetic field effects on human lymphocytes

The results are discussed of a systematic investigation into the electromagnetic field (EMP) exposure consequences on human lymphocytes. These artificial fields have intensities comparable to the Earths magnetic field, and are used for exposures up to 4 days. Different and complementary techniques are used to safely assess the consequences of EMFs on the cells; in particular, morphology, metabolism, and population dynamics are investigated. The recourse to ultramicroscopy, pressure monitoring in sealed bottles, atomic mass spectroscopy, and cytofluorimetry techniques give good insight into the EMF-induced changes. A statistically significant deviation of irradiated samples with respect to control samples is reported. A critical analysis and a survey of similar experiments reported in the literature led us to examine the experimental setup with attention to the geometry of the irradiation system. Yeast cells were used as a model system to statistically test the different steps in the overall procedure, thanks to information gathered during a radiobiology experiment performed at the Rutherford Appleton Laboratory. Finally, the role of different magnetic field detectors in the reproducibility of the experiments is carefully discussed.

Hydrogen peroxide mechanosynthesis in siloxane-hydrogel contact lenses

Drug-loaded contact lenses are emerging as the preferred treatment method for several ocular diseases, and efforts are being directed to promote extended and controlled delivery. One strategy is based on delivery induced by environmental triggers. One of these triggers can be hydrogen peroxide, since many platforms based on drug-loaded nanoparticles were demonstrated to be hydrogen-peroxide responsive. This is particularly interesting when hydrogen peroxide is the result of a specific pathophysiological condition. Otherwise, an alternative route to induce drug delivery is here proposed, namely the mechano-synthesis. The present work represents the proof-of-concept of the mechanosynthesis of hydrogen peroxide in siloxane-hydrogel contact lenses as a consequence of the cleavage of siloxane bonds at the interface between the polymer and water in aqueous phase. Their spongy morphology makes contact lenses promising systems for mechanical-to-chemical energy conversion, since the amount of hydrogen peroxide is expected to scale with the interfacial area between the polymer and water. The eyelid pressure during wear is sufficient to induce the hydrogen peroxide synthesis with concentrations which are biocompatible and suitable to trigger the drug release through hydrogen-peroxide-responsive platforms. For possible delivery on demand, the integration of piezoelectric polymers in the siloxane-hydrogel contact lenses could be designed, whose mechanical deformation could be induced by an applied wireless-controlled voltage.

An ensemble of new techniques to study soft-X-ray-induced variations in cellular metabolism

An ensemble of new techniques has been developed to study cell metabolism. These include: CO2 production monitoring, cell irradiation with soft X rays produced with a laser-plasma source, and study of oscillations in cell metabolic activity via spectral analysis of experimental records. Soft X-rays at about 0.9 keV, with a very low penetration in biological material, were chosen to produce damages at the metabolic level, without great interference with DNA activity. The use of a laser-plasma source allowed a fast deposition of high doses. Monitoring of CO2 production allowed us to measure cell metabolic response immediately after irradiation in a continuous and non invasive way. Also a simple model was developed to calculate X-ray doses delivered to the different cell compartments following a Lambert-Bouguet–Beer law. Results obtained on Saccharomyces cerevisiae yeast cells in experiments performed at Rutherford Appleton Laboratory are presented.

Magnetic field effects on human lymphocytes : methodological assessments and experimental evidences

The results are discussed of a systematic investigation on the electromagnetic field exposure consequences on human lymphocytes. These artificial fields have intensities comparable with the Earth magnetic field one, and are used for exposures up to 4 days. Different and complementary techniques are used to safely assess the consequences of ElectroMagnetic Fields (EMF) on the cells; in particular morphology, metabolism and population dynamics are investigated. The recourse to ultra microscopy, pressure monitoring in sealed bottles, atomic mass spectroscopy. Far IR Fourier Transform and cytofluorimetry techniques give a good insight in the EMF induced changes. A statistically significant deviation of irradiated samples with respect to the control ones are reported. A critical analysis and a survey of similar experiments reported in literature lead us to the exam of the experimental set up with attention to the geometry of the irradiation system. Finally the role of different magnetic field detectors in the reproducibility of the experiments will be carefully discussed.

Water-Assisted Hole Trapping at the Highly Curved Surface of Nano-TiO2 Photocatalyst

Heterogeneous photocatalysis is vital in solving energy and environmental issues that this society is confronted with. Although photocatalysts are often operated in the presence of water, it has not been yet clarified how the interaction with water itself affects charge dynamics in photocatalysts. Using water-coverage-controlled steady and transient infrared absorption spectroscopy and large-model (∼800 atoms) ab initio calculations, we clarify that water enhances hole trapping at the surface of TiO2 nanospheres but not of well-faceted nanoparticles. This water-assisted effect unique to the nanospheres originates from water adsorption as a ligand at a low-coordinated Ti–OH site or through robust hydrogen bonding directly to the terminal OH at the highly curved nanosphere surface. Thus, the interaction with water at the surface of nanospheres can promote photocatalytic reactions of both oxidation and reduction by elongating photogenerated carrier lifetimes. This morphology-dependent water-assisted effect provides a novel and rational basis for designing and engineering nanophotocatalyst morphology to improve photocatalytic performances.

Morphometric Analyses by a New Slit-lamp Endothelial Biomicroscope

Purpose: A method called EndoKer was recently devised for the morphometric analysis of the cell mosaic of the corneal endothelium. Fully automatic cell recognition is performed on images acquired by a slit-lamp biomicroscope. The aim of this study was to evaluate the accuracy of the EndoKer results. Methods: Analyses were performed on a polystyrene bead layer stratified on a contact lens and in vivo on 30 adults. Accuracy was evaluated by comparing the results of EndoKer with the true values obtained by manual counting of the cells in the same images. EndoKer results were also compared with those obtained with the Tomey EM3000 microscope. Results: The accuracy of the results compared with the manual counting on the same images showed a difference of a few percent for the cell density and for hexagonality. This high accuracy derives from (1) the resolution of the slit-lamp images and (2) the improved cell recognition of the fully automatic method. A good agreement was also found between EndoKer and the Tomey EM3000 microscope results. Conclusions: Based on the investigated 30 cases, the slit-lamp biomicroscope may be a viable alternative to dedicated endothelial instruments, providing the additional advantages of a larger investigated area and the possibility to take images of different portions of the cornea. The calibration was performed during the development of the method by using polystyrene beads. The user is not required to perform this calibration. However, such a calibrated sample is suggested for those interested.

Mechanically triggered solute uptake in soft contact lenses

Molecular arrangement plays a role in the diffusion of water and solutes across soft contact lenses. In particular, the uptake of solutes in hydrated contact lenses can occur as long as free water is available for diffusion. In this work, we investigated the effect of mechanical vibrations of low frequency (200 Hz) on the solute uptake. Hyaluronan, a polysaccharide of ophthalmic use, was taken as example of solute of interest. For a specific water-hydrated hydrogel material, differential scanning calorimetry experiments showed that a large fraction of the hydration water accounted for loosely-bound water, both before and after one week of daily-wear of the lenses. The size (of the order of magnitude of few hundreds of nanometers) of hyaluronan in aqueous solution was found to be less than the size of the pores of the lens observed by scanning electron microscopy. However, solute uptake in already-hydrated lenses was negligible by simple immersion, while a significant increase occurred under mechanical vibrations of 200 Hz, thus providing experimental evidence of mechanically triggered enhanced solute uptake, which is attributed to the release of interfacial loosely-bound water. Also other materials were taken into consideration. However, the effectiveness of mechanical vibrations for hyaluronan uptake is restricted to lenses containing interfacial loosely-bound water. Indeed, loosely-bound water is expected to be bound to the polymer with bonding energies of the order of magnitude of 10-100 J/g, which are compatible with the energy input supplied by the vibrations.

Care System Versus Transmitted Light Wavefront Pattern of Contact Lenses.

Objectives: This article compares the optical performance of soft contact lenses (CLs) treated with multipurpose or hydrogen peroxide care systems. Methods: The investigated care systems were (1) 3% hydrogen peroxide solution Oxysept (Abbot Medical Optics, Abbott Park, IL) and (2) multipurpose solution Regard (Vita Research, Ariccia, Italy). Three types of silicone hydrogel CLs were studied (comfilcon A, lotrafilcon B, and balafilcon A), unworn and exposed for 30 times to the solutions, which were replaced every 8 hr. The optical performance of the CLs was evaluated through the on-eye transmitted light wavefront patterns by considering new CLs as references. The surface morphology of the CLs was investigated by scanning electron microscopy. Results: Statistically significant modifications in the range 0.1 to 0.3 &mgr;m of Zernicke coefficients and modifications of the root mean square of the wavefront aberration function were found for CLs treated with multipurpose solution, in agreement with the observed modifications of the surface morphology. Statistically significant changes were also found after exposure to the hydrogen peroxide solution, but the variation of the Zernicke coefficients was found lower than 0.1 &mgr;m, thus being negligible in CL optical performances. Conclusions: In addition to disinfection ability and ocular surface reactions, CL care systems are different in solution-related CL optical performance. Multipurpose solutions may affect the CL surface morphology with significant modifications of the transmitted light wavefront pattern.

Variable-Size Bead Layer as Standard Reference for Endothelial Microscopes

PURPOSE For morphometric analysis of the cell mosaic of corneal endothelium, checking accuracy and precision of instrumentation is a key step. In this study, a standard reference sample is proposed, developed to reproduce the cornea with its shape and the endothelium with its intrinsic variability in the cell size. METHODS A polystyrene bead layer (representing the endothelium) was deposited on a lens (representing the cornea). Bead diameters were 20, 25, and 30 μm (fractions in number 55%, 30%, and 15%, respectively). Bead density and hexagonality were simulated to obtain the expected true values and measured using a slit-lamp endothelial microscope applied to 1) a Takagi 700GL slit lamp at 40× magnification (recommended standard setup) and 2) a Takagi 2ZL slit lamp at 25× magnification. RESULTS The simulation provided the expected bead density 2001 mm and hexagonality 47%. At 40×, density and hexagonality were measured to be 2009 mm (SD 93 mm) and 45% (SD 3%). At 25× on a different slit lamp, the comparison between measured and expected densities provided the factor 1.526 to resize the image and to use the current algorithms of the slit-lamp endothelial microscope for cell recognition. CONCLUSIONS A variable-size polystyrene bead layer on a lens is proposed as a standard sample mimicking the real shape of the cornea and the variability of cell size and cell arrangement of corneal endothelium. The sample is suggested to evaluate accuracy and precision of cell density and hexagonality obtained by different endothelial microscopes, including a slit-lamp endothelial microscope applied to different slit lamps, also at different magnifications.PURPOSE For morphometric analysis of the cell mosaic of corneal endothelium, checking accuracy and precision of instrumentation is a key step. In this study, a standard reference sample is proposed, developed to reproduce the cornea with its shape and the endothelium with its intrinsic variability in the cell size. METHODS A polystyrene bead layer (representing the endothelium) was deposited on a lens (representing the cornea). Bead diameters were 20, 25, and 30 μm (fractions in number 55%, 30%, and 15%, respectively). Bead density and hexagonality were simulated to obtain the expected true values and measured using a slit-lamp endothelial microscope applied to 1) a Takagi 700GL slit lamp at 40× magnification (recommended standard setup) and 2) a Takagi 2ZL slit lamp at 25× magnification. RESULTS The simulation provided the expected bead density 2001 mm and hexagonality 47%. At 40×, density and hexagonality were measured to be 2009 mm (SD 93 mm) and 45% (SD 3%). At 25× on a different slit lamp, the comparison between measured and expected densities provided the factor 1.526 to resize the image and to use the current algorithms of the slit-lamp endothelial microscope for cell recognition. CONCLUSIONS A variable-size polystyrene bead layer on a lens is proposed as a standard sample mimicking the real shape of the cornea and the variability of cell size and cell arrangement of corneal endothelium. The sample is suggested to evaluate accuracy and precision of cell density and hexagonality obtained by different endothelial microscopes, including a slit-lamp endothelial microscope applied to different slit lamps, also at different magnifications.