Cristina Lenardi

Quantitative Characterization of the Influence of the Nanoscale Morphology of Nanostructured Surfaces on Bacterial Adhesion and Biofilm Formation

Bacterial infection of implants and prosthetic devices is one of the most common causes of implant failure. The nanostructured surface of biocompatible materials strongly influences the adhesion and proliferation of mammalian cells on solid substrates. The observation of this phenomenon has led to an increased effort to develop new strategies to prevent bacterial adhesion and biofilm formation, primarily through nanoengineering the topology of the materials used in implantable devices. While several studies have demonstrated the influence of nanoscale surface morphology on prokaryotic cell attachment, none have provided a quantitative understanding of this phenomenon. Using supersonic cluster beam deposition, we produced nanostructured titania thin films with controlled and reproducible nanoscale morphology respectively. We characterized the surface morphology; composition and wettability by means of atomic force microscopy, X-ray photoemission spectroscopy and contact angle measurements. We studied how protein adsorption is influenced by the physico-chemical surface parameters. Lastly, we characterized Escherichia coli and Staphylococcus aureus adhesion on nanostructured titania surfaces. Our results show that the increase in surface pore aspect ratio and volume, related to the increase of surface roughness, improves protein adsorption, which in turn downplays bacterial adhesion and biofilm formation. As roughness increases up to about 20 nm, bacterial adhesion and biofilm formation are enhanced; the further increase of roughness causes a significant decrease of bacterial adhesion and inhibits biofilm formation. We interpret the observed trend in bacterial adhesion as the combined effect of passivation and flattening effects induced by morphology-dependent protein adsorption. Our findings demonstrate that bacterial adhesion and biofilm formation on nanostructured titanium oxide surfaces are significantly influenced by nanoscale morphological features. The quantitative information, provided by this study about the relation between surface nanoscale morphology and bacterial adhesion points towards the rational design of implant surfaces that control or inhibit bacterial adhesion and biofilm formation.

Evidence of Extended Solidlike Layering in [Bmim][NTf2] Ionic Liquid Thin Films at Room-Temperature

We report the direct observation of solidlike ordering at room temperature of thin films of [Bmim][NTf2] ionic liquid on mica, amorphous silica, and oxidized Si(110). A statistical quantitative analysis of atomic force microscopy topographies shows that on these surfaces [Bmim][NTf2] forms layered structures, characterized by a perpendicular structural periodicity of approximately 0.6 nm. Remarkably, even the highest structures, up to 50 nm high, behave solidlike against the AFM probe. Conversely, on highly oriented pyrolitic graphite the ionic liquid forms nanometer-sized, liquidlike domains. The results of this study are directly relevant for those applications where ILs are employed in form of thin films supported on solid surfaces, such as in microelectromechanical or microelectronic devices. More generally, they suggest that at the liquid/solid interface the structural properties of ILs can be far more complex than those depicted so far, and prompt new fundamental investigations of the forces that drive supported ILs through a liquidlike-to-solidlike transition.

The influence of the precursor clusters on the structural and morphological evolution of nanostructured TiO2 under thermal annealing

We have produced nanostructured titanium dioxide thin films by supersonic cluster beam deposition. The as-deposited films have a nanocrystalline or amorphous structure depending on the mass distribution of the precursor clusters. This can be controlled by aerodynamic separation effects typical of supersonic expansions. On thermal annealing at temperatures from 400 to 800 °C in ambient atmosphere, amorphous-to-anatase and anatase-to-rutile phase transitions have been observed. The nanostructure and microstructure evolution of the film upon annealing has been characterized by atomic force microscopy and transmission electron microscopy. The influence of the precursor clusters in the evolution of the film nanostructure at high temperatures has been demonstrated. This observation opens up new perspectives for batch fabrication of devices based on cluster-assembled materials.

Fabrication of Polymer Microneedles Using a Two-Photon Polymerization and Micromolding Process

Background: Microneedle-mediated drug delivery is a promising method for transdermal delivery of insulin, incretin mimetics, and other protein-based pharmacologic agents for treatment of diabetes mellitus. One factor that has limited clinical application of conventional microneedle technology is the poor fracture behavior of microneedles that are created using conventional materials and methods. In this study polymer microneedles for transdermal delivery were created using a two-photon polymerization (2PP) microfabrication and subsequent polydimethylsiloxane (PDMS) micromolding process. Methods: Solid microneedle arrays, fabricated by means of 2PP, were used to create negative molds from PDMS. Using these molds microneedle arrays were subsequently prepared by molding eShell 200, a photo-reactive acrylate-based polymer that exhibits water and perspiration resistance. Results: The eShell 200 microneedle array demonstrated suitable compressive strength for use in transdermal drug delivery applications. Human epidermal keratinocyte viability on the eShell 200 polymer surfaces was similar to that on polystyrene control surfaces. In vitro studies demonstrated that eShell 200 microneedle arrays fabricated using the 2PP microfabrication and PDMS micromolding process technique successfully penetrated human stratum corneum and epidermis. Conclusions: Our results suggest that a 2PP microfabrication and subsequent PDMS micromolding process may be used to create microneedle structures with appropriate structural, mechanical, and biological properties for transdermal drug delivery of insulin and other protein-based pharmacologic agents for treatment of diabetes mellitus.

Versatile fabrication of vascularizable scaffolds for large tissue engineering in bioreactor

Despite significant progresses were achieved in tissue engineering over the last 20 years, a number of unsolved problems still remain. One of the most relevant issues is the lack of a proper vascularization that is limiting the size of the engineered tissues to smaller than clinically relevant dimensions. Sacrificial molding holds great promise to engineered construct with perfusable vascular architectures, but there is still the need to develop more versatile approaches able to be independent of the nature and dimensions of the construct. In this work we developed a versatile sacrificial molding technique for fabricating bulk, cell-laden and porous scaffolds with embedded vascular fluidic networks. These branched fluidic architectures are created by highly resistant thermoplastic sacrificial templates, made of poly(vinyl alcohol), representing a remarkable progress in manufacturability and scalability. The obtained architecture, when perfused in bioreactor, has shown to prevent the formation of a necrotic core in thick cell-laden constructs and enabled the rapid fabrication of hierarchically branched endothelium. In conclusion we demonstrate a novel strategy towards the engineering of vascularized thick tissues through the integration of the PVA-based microfabrication sacrificial approach and perfusion bioreactors. This approach may be able to scale current engineered tissues to clinically relevant dimensions, opening the way to their widespread clinical applications.

Cluster beam synthesis of nanostructured thin films

The use of clusters as elemental building blocks can open routes toward the fabrication of a new class of nanostructured solids and devices. We report the synthesis of nanostructured films using supersonic cluster beam deposition. A new type of cluster source based on microplasma ablation has been developed. This allows a substantial improvement in terms of deposition rate and control on cluster mass distribution. These achievements make supersonic cluster beams a useful tool in the arena of cluster assembling of materials. We have applied this technique to the growth of nanostructured carbon thin films. The structure and morphology of the films can be controlled by varying the cluster mass distribution prior to deposition. Deposition conditions affect the surface roughness and the onset of scale-invariant morphology on a dimension domain extending from the nanometer up to the micrometer. The cluster beam deposition method shows very promising features in view of the large scale growth of nanostructured f...

A comparative study between AFM and SEM imaging on human scalp hair

A comparative study of AFM and SEM imaging of the same area of a human scalp hair has been carried out to determine the similarity and the differences between the two techniques. Sample preparation for SEM analysis requires a metallization step and vacuum exposure, both of which could potentially induce modifications to the surface details. By contrast, AFM is a suitable technique to evaluate any effect resulting from sample manipulation because it can be applied without any specific treatment. AFM analysis demonstrates that sample metallization is responsible for modifications to the surface details of hair, mainly comprising an increase in height of scale steps and of root mean square roughness together with variation in scale profiles. Sample treatments for SEM imaging are in general potentially responsible for surface modifications to the samples involved.

Three-dimensional hypoxic culture of human mesenchymal stem cells encapsulated in a photocurable, biodegradable polymer hydrogel: a potential injectable cellular product for nucleus pulposus regeneration

Nucleus pulposus (NP) tissue damage can induce detrimental mechanical stresses and strains on the intervertebral disc, leading to disc degeneration. This study demonstrates the potential of a novel, photo-curable, injectable, synthetic polymer hydrogel (pHEMA-co-APMA grafted with polyamidoamine (PAA)) to encapsulate and differentiate human mesenchymal stem cells (hMSC) towards a NP phenotype under hypoxic conditions which could be used to restore NP tissue function and mechanical properties. Encapsulated hMSC cultured in media (hMSC and chondrogenic) displayed good cell viability up to day 14. The genotoxicity effects of ultraviolet (UV) on hMSC activity confirmed the acceptability of 2.5min of UV light exposure to cells. Cytotoxicity investigations revealed that hMSC cultured in media containing p(HEMA-co-APMA) grafted with PAA degradation product (10% and 20%v/v concentration) for 14days significantly decreased the initial hMSC adhesion ability and proliferation rate from 24hrs to day 14. Successful differentiation of encapsulated hMSC within hydrogels towards chondrogenesis was observed with elevated expression levels of aggrecan and collagen II when cultured in chondrogenic media under hypoxic conditions, in comparison with culture in hMSC media for 14days. Characterization of the mechanical properties revealed a significant decrease in stiffness and modulus values of cellular hydrogels in comparison with acellular hydrogels at both day 7 and day 14. These results demonstrate the potential use of an in vivo photo-curable injectable, synthetic hydrogel with encapsulated hMSC for application in the repair and regeneration of NP tissue.

Direct Microfabrication of Topographical and Chemical Cues for the Guided Growth of Neural Cell Networks on Polyamidoamine Hydrogels

Cell patterning is an important tool for organizing cells in surfaces and to reproduce in a simple way the tissue hierarchy and complexity of pluri-cellular life. The control of cell growth, proliferation and differentiation on solid surfaces is consequently important for prosthetics, biosensors, cell-based arrays, stem cell therapy and cell-based drug discovery concepts. We present a new electron beam lithography method for the direct and simultaneous fabrication of sub-micron topographical and chemical patterns, on a biocompatible and biodegradable PAA hydrogel. The localized e-beam modification of a hydrogel surface makes the pattern able to adsorb proteins in contrast with the anti-fouling surface. By also exploiting the selective attachment, growth and differentiation of PC12 cells, we fabricated a neural network of single cells connected by neuritis extending along microchannels. E-beam microlithography on PAA hydrogels opens up the opportunity of producing multifunctional microdevices incorporating complex topographies, allowing precise control of the growth and organization of individual cells.

NEXAFS characterization of nanostructured carbon thin-films exposed to hydrogen

Abstract Nanostructured carbon films were grown by supersonic cluster beam deposition using a focusing nozzle for selecting small clusters and increasing the deposition rate. The films present a pore structure in the mesopore range (maximum of the pore size distribution at 34 A) with a specific surface area of 665 m 2 /g. Temperature programmed desorption (TPD) analysis shows sorption of hydrogen after hydrogen exposure. In the near edge X-ray absorption fine structure (NEXAFS) spectra a strong peak at ∼288.5 eV, related to C–H bonds (hydrogen chemisorbed), is evident. After sample annealing at temperature of approximately 500°C, the prominent C–H* peak is strongly reduced. The samples experienced reiterated exposures to water vapor and hydrogen and subsequent annealings. The partial restoration of the C–H* peak after exposures and its decrease after moderate heating indicate that the sorption and release of hydrogen is a reversible process.