Design and fabrication of biocompatible scaffolds for the regeneration of tissues

Regenerative medicine and tissue engineering attempt to repair or improve the biological functions of tissues that have been damaged or have ceased to perform their role through three main components: a biocompatible scaffold, cellular component and bioactive molecules. Nanotechnology provide a toolbox of innovative scaffold fabrication procedures in regenerative medicine. In fact, nanotechnology, using manufacturing techniques such as conventional and unconventional lithography, allows fabricating supports with different geometries and sizes as well as displaying physical chemical properties tunable over different length scales. Soft lithography techniques allow to functionalize the support by specific molecules that promote adhesion and control the growth of cells. Understanding cell response to scaffold, and viceversa, is a key issue; here we show our investigation of the essential features required for improving the cell-surface interaction over different scale lengths. The main goal of this thesis has been to devise a nanotechnology-based strategy for the fabrication of scaffolds for tissue regeneration. We made four types of scaffolds, which are able to accurately control cell adhesion and proliferation. For each scaffold, we chose properly designed materials, fabrication and characterization techniques

Biological niches within human calcified aortic valves. Towards understanding of the pathological biomineralization process

Despite recent advances, mineralization site, its microarchitecture, and composition in calcific heart valve remain poorly understood. A multiscale investigation, using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy dispersive X-ray spectrometry (EDS), from micrometre up to nanometre, was conducted on human severely calcified aortic and mitral valves, to provide new insights into calcificationp rocess. Our aim was to evaluate the spatial relationship existing between bioapatite crystals, their local growing microenvironment, and the presence of a hierarchical architecture. Here we detected the presence of bioapatite crystals in two different mineralization sites that suggest the action of two different growth processes:a pathological crystallization process that occurs in biological niches and is ascribed to a purely physicochemical process and a matrix- mediated mineralized process in which the extracellular matrix acts as the template for a site-directed nanocrystals nucleation. Different shapes of bioapatite crystallization were observed at micrometer scale in each microenvironment but at the nanoscale level crystals appear to be made up by the same subunit

Multiscale Investigation of Random Heterogenous Media in Materials and Earth Sciences

This dissertation is concerned with three major areas pertaining to the characterization and analysis of heterogeneous materials. The first is focused on the modeling of heterogeneous materials with random microstructure and understanding their thermomechanical properties as well as developing a methodology for the multiscale thermoelastic analysis of random heterogeneous materials. Realistic random microstructures are generated for computational analyses using random morphology description functions. The simulated microstructures closely resemble actual micrographs of random heterogeneous materials. The simulated random microstructures are characterized using statistical techniques and their homogenized material properties computed using the asymptotic expansion homogenization method. The failure response of random media is investigated via a direct micromechanical failure analysis which utilizes stresses at the microstructural level coupled with appropriate phase material failure models to generate initial failure envelopes. The homogenized material properties and failure envelopes are employed to perform accurate coupled macroscale and microscale analyses of random heterogeneous material components. The second area addressed in this dissertation involves the transient multiscale analysis of two-phase functionally graded materials within the framework of linearized thermoelasticity. The two-phase material microstructures, which are created using a morphology description function, have smoothly varying microstructure morphologies that depend on the volume fractions of the constituent phases. The multiscale problem is analyzed using asymptotic expansion homogenization coupled with the finite element method. Model problems are studied to illustrate the versatility of the multiscale analysis procedure which incorporates a direct micromechanical failure analysis to accurately compute the factors of safety for functionally graded components. The last area of this dissertation is concerned with determining the role of heterogeneous rock fabric features in quartz/muscovite rich rocks on seismic wave speed anisotropy. The bulk elastic properties and corresponding wave velocities are calculated for synthetic heterogeneous rock microstructures with varying material and geometric features to investigate their influence on seismic wave speed anisotropy. The asymptotic expansion homogenization method is employed to calculate precise bulk stiffness tensors for representative rock volumes and the wave speed velocities are obtained from the Christoffel equation. The obtained results are also used to assess the performance of analytic homogenization schemes currently used in the geophysics community

Tuning the plasmonic properties of silver nanopatterns fabricated by shadow nanosphere lithography

Regular silver (Ag) nanopatterns, from disconnected nanotriangles to well coupled triangular clusters of nanoparticles, were prepared by shadow nanosphere lithography at different incident angles θ from 0 degrees to 20 degrees with continuous azimuthal rotation. The resulting nanopatterns were consistent with predictions by numerical calculations and Monte Carlo simulations of adatoms with high diffusivity. The visible localized surface plasmon resonance of these nanopatterns was tuned by θ systematically due to the change in size, shape, and arrangement of Ag nanopatterns. These resonances were consistent with finite-difference time-domain simulations using realistic nanopatterns based upon scanning electron micrographs. Such a simple fabrication strategy can be used to optimize surface enhanced Raman scattering substrate fabrication, as well as other plasmonics based applications

Automated segmentation of tissue images for computerized IHC analysis

This paper presents two automated methods for the segmentation ofimmunohistochemical tissue images that overcome the limitations of themanual approach aswell as of the existing computerized techniques. The first independent method, based on unsupervised color clustering, recognizes automatically the target cancerous areas in the specimen and disregards the stroma; the second method, based on colors separation and morphological processing, exploits automated segmentation of the nuclear membranes of the cancerous cells. Extensive experimental results on real tissue images demonstrate the accuracy of our techniques compared to manual segmentations; additional experiments show that our techniques are more effective in immunohistochemical images than popular approaches based on supervised learning or active contours. The proposed procedure can be exploited for any applications that require tissues and cells exploration and to perform reliable and standardized measures of the activity of specific proteins involved in multi-factorial genetic pathologie

Reconstituting ring-rafts in bud-mimicking topography of model membranes.

During vesicular trafficking and release of enveloped viruses, the budding and fission processes dynamically remodel the donor cell membrane in a protein- or a lipid-mediated manner. In all cases, in addition to the generation or relief of the curvature stress, the buds recruit specific lipids and proteins from the donor membrane through restricted diffusion for the development of a ring-type raft domain of closed topology. Here, by reconstituting the bud topography in a model membrane, we demonstrate the preferential localization of cholesterol- and sphingomyelin-enriched microdomains in the collar band of the bud-neck interfaced with the donor membrane. The geometrical approach to the recapitulation of the dynamic membrane reorganization, resulting from the local radii of curvatures from nanometre-to-micrometre scales, offers important clues for understanding the active roles of the bud topography in the sorting and migration machinery of key signalling proteins involved in membrane budding

Non-equilibrium melting of colloidal crystals in confinement

We report on a novel and flexible experiment to investigate the non-equilibrium melting behaviour of model crystals made from charged colloidal spheres. In a slit geometry polycrystalline material formed in a low salt region is driven by hydrostatic pressure up an evolving gradient in salt concentration and melts at large salt concentration. Depending on particle and initial salt concentration, driving velocity and the local salt concentration complex morphologic evolution is observed. Crystal-melt interface positions and the melting velocity are obtained quantitatively from time resolved Bragg- and polarization microscopic measurements. A simple theoretical model predicts the interface to first advance, then for balanced drift and melting velocities to become stationary at a salt concentration larger than the equilibrium melting concentration. It also describes the relaxation of the interface to its equilibrium position in a stationary gradient after stopping the drive in different manners. We further discuss the influence of the gradient strength on the resulting interface morphology and a shear induced morphologic transition from polycrystalline to oriented single crystalline material before melting

Multiscale reinforcing interlayers of self-same P(St-co-GMA) nanofibers loaded with MCF for polymer composites and nanocomposites

Electrospinning has become a proven technique to introduce polymeric sub-phases into composites. The sub-phases such as nanofibers can also be used as a carrier platform for reinforcing particles at different scales, enabling a multiscale reinforcement approach. However, the polymeric nanofibers may lose their intended fibrous morphology during the composite curing at elevated temperature. As such, polymeric sub-phase can not contribute effectively as fibers to the mechanical properties of the composite. This paper exemplifies introduction of milled carbon fibers (MCF) carried by electrospun polymeric nanofibers and the use of the resultant multi-scale reinforcement as interlayer within conventional structural composites. The issue of polymeric nanofibers exposed to elevated temperature curing is circumvented by implementing a novel self-same nanofibrous strategy. While a base polymer for the nanofibers is chosen as epoxy compatible P(St-co-GMA), its derivative by a cross-linker Phthalic Anhydrate, P(St-co-GMA)/PA is also incorporated by dual-electrospining, i.e. simultaneous electrospinning of the two polymers. It was shown that the nanofibers of the base polymer melt and fuse over the cross-linkable nanofibers forming the self-same nanofibrous morphology during the heat treatment in accordance with the cure cycle of the epoxy resin in this study. MCFs were mixed into the cross-linkable polymer solution and electrospun with the P(St-co-GMA)/PA nanofibers. The dual polymer and MCF loaded nanofibrous structures were analyzed morphologically before and after heat treatment. Homogenous distribution of particles in the fibrous structures, melting of the neat copolymer, crosslinking of the polymer mix, and selfsame fibrous structure were characterized. The nanofiber mats were used as the reinforcement to epoxy resin films and as interlayers for carbon fiber-reinforced composites. In the case of nanocomposites, MCF enhanced the elastic modulus by about 9%. In the use of multiscale nanofibrous mats as interlayers of continuous carbon fiber composites, they improved the ultimate tensile strength of a cross-ply laminate by 9%