Porous Polymeric Bioresorbable Scaffolds for Tissue Engineering

Tissue engineering is a discipline that aims at regenerating damaged biological tissues by using a cell-construct engineered in vitro made of cells grown into a porous 3D scaffold. The role of the scaffold is to guide cell growth and differentiation by acting as a bioresorbable temporary substrate that will be eventually replaced by new tissue produced by cells. As a matter or fact, the obtainment of a successful engineered tissue requires a multidisciplinary approach that must integrate the basic principles of biology, engineering and material science. The present Ph.D. thesis aimed at developing and characterizing innovative polymeric bioresorbable scaffolds made of hydrolysable polyesters. The potentialities of both commercial polyesters (i.e. poly-e-caprolactone, polylactide and some lactide copolymers) and of non-commercial polyesters (i.e. poly-w-pentadecalactone and some of its copolymers) were explored and discussed. Two techniques were employed to fabricate scaffolds: supercritical carbon dioxide (scCO2) foaming and electrospinning (ES). The former is a powerful technology that enables to produce 3D microporous foams by avoiding the use of solvents that can be toxic to mammalian cells. The scCO2 process, which is commonly applied to amorphous polymers, was successfully modified to foam a highly crystalline poly(w-pentadecalactone-co-e-caprolactone) copolymer and the effect of process parameters on scaffold morphology and thermo-mechanical properties was investigated. In the course of the present research activity, sub-micrometric fibrous non-woven meshes were produced using ES technology. Electrospun materials are considered highly promising scaffolds because they resemble the 3D organization of native extra cellular matrix. A careful control of process parameters allowed to fabricate defect-free fibres with diameters ranging from hundreds of nanometers to several microns, having either smooth or porous surface. Moreover, versatility of ES technology enabled to produce electrospun scaffolds from different polyesters as well as “composite” non-woven meshes by concomitantly electrospinning different fibres in terms of both fibre morphology and polymer material. The 3D-architecture of the electrospun scaffolds fabricated in this research was controlled in terms of mutual fibre orientation by properly modifying the instrumental apparatus. This aspect is particularly interesting since the micro/nano-architecture of the scaffold is known to affect cell behaviour. Since last generation scaffolds are expected to induce specific cell response, the present research activity also explored the possibility to produce electrospun scaffolds bioactive towards cells. Bio-functionalized substrates were obtained by loading polymer fibres with growth factors (i.e. biomolecules that elicit specific cell behaviour) and it was demonstrated that, despite the high voltages applied during electrospinning, the growth factor retains its biological activity once released from the fibres upon contact with cell culture medium. A second fuctionalization approach aiming, at a final stage, at controlling cell adhesion on electrospun scaffolds, consisted in covering fibre surface with highly hydrophilic polymer brushes of glycerol monomethacrylate synthesized by Atom Transfer Radical Polymerization. Future investigations are going to exploit the hydroxyl groups of the polymer brushes for functionalizing the fibre surface with desired biomolecules. Electrospun scaffolds were employed in cell culture experiments performed in collaboration with biochemical laboratories aimed at evaluating the biocompatibility of new electrospun polymers and at investigating the effect of fibre orientation on cell behaviour. Moreover, at a preliminary stage, electrospun scaffolds were also cultured with tumour mammalian cells for developing in vitro tumour models aimed at better understanding the role of natural ECM on tumour malignity in vivo.

Biomimetic Scaffolds for the Controlled Release of Bioactive Molecules for Tissue Engineering Applications

The temporospatial controlled delivery of growth factors is crucial to trigger the desired healing mechanisms in target tissues. The uncontrolled release of growth factors has been demonstrated to cause severe side effects in its surrounding tissues. Thus, the first working hypothesis was to tune and optimize a newly developed multiscale delivery platform based on a nanostructured silicon particle core (pSi) and a poly (dl-lactide-co-glycolide) acid (PLGA) outer shell. In a murine subcutaneous model, the platform was demonstrated to be fully tunable for the temporal and spatial control release of the payload. Secondly, a multiscale approach was followed in a multicompartment collagen scaffold, to selectively integrate different sets of PLGA-pSi loaded with different reporter proteins. The spatial confinement of the microspheres allowed the release of the reporter proteins in each of the layers of the scaffold. Finally, the staged and zero-order release kinetics enabled the temporal biochemical patterning of the scaffold. The last step of this PhD project was to test if by fully embedding PLGA microspheres in a highly structured and fibrous collagen-based scaffold (camouflaging), it was possible to prevent their early detection and clearance by macrophages. It was further studied whether such a camouflaging strategy was efficient in reducing the production of key inflammatory molecules, while preserving the release kinetics of the payload of the PLGA microspheres. Results demonstrated that the camouflaging allowed for a 10-fold decrease in the number of PLGA microspheres internalized by macrophages, suggesting that the 3D scaffold operated by cloaking the PLGA microspheres. When the production of key inflammatory cytokines induced by the scaffold was assessed, macrophages' response to the PLGA microspheres-integrated scaffolds resulted in a response similar to that observed in the control (not functionalized scaffold) and the release kinetic of a reporter protein was preserved.

Meta-models, environment and layers: agent-oriented engineering of complex systems

Traditional software engineering approaches and metaphors fall short when applied to areas of growing relevance such as electronic commerce, enterprise resource planning, and mobile computing: such areas, in fact, generally call for open architectures that may evolve dynamically over time so as to accommodate new components and meet new requirements. This is probably one of the main reasons that the agent metaphor and the agent-oriented paradigm are gaining momentum in these areas. This thesis deals with the engineering of complex software systems in terms of the agent paradigm. This paradigm is based on the notions of agent and systems of interacting agents as fundamental abstractions for designing, developing and managing at runtime typically distributed software systems. However, today the engineer often works with technologies that do not support the abstractions used in the design of the systems. For this reason the research on methodologies becomes the basic point in the scientific activity. Currently most agent-oriented methodologies are supported by small teams of academic researchers, and as a result, most of them are in an early stage and still in the first context of mostly \academic" approaches for agent-oriented systems development. Moreover, such methodologies are not well documented and very often defined and presented only by focusing on specific aspects of the methodology. The role played by meta- models becomes fundamental for comparing and evaluating the methodologies. In fact a meta-model specifies the concepts, rules and relationships used to define methodologies. Although it is possible to describe a methodology without an explicit meta-model, formalising the underpinning ideas of the methodology in question is valuable when checking its consistency or planning extensions or modifications. A good meta-model must address all the different aspects of a methodology, i.e. the process to be followed, the work products to be generated and those responsible for making all this happen. In turn, specifying the work products that must be developed implies dening the basic modelling building blocks from which they are built. As a building block, the agent abstraction alone is not enough to fully model all the aspects related to multi-agent systems in a natural way. In particular, different perspectives exist on the role that environment plays within agent systems: however, it is clear at least that all non-agent elements of a multi-agent system are typically considered to be part of the multi-agent system environment. The key role of environment as a first-class abstraction in the engineering of multi-agent system is today generally acknowledged in the multi-agent system community, so environment should be explicitly accounted for in the engineering of multi-agent system, working as a new design dimension for agent-oriented methodologies. At least two main ingredients shape the environment: environment abstractions - entities of the environment encapsulating some functions -, and topology abstractions - entities of environment that represent the (either logical or physical) spatial structure. In addition, the engineering of non-trivial multi-agent systems requires principles and mechanisms for supporting the management of the system representation complexity. These principles lead to the adoption of a multi-layered description, which could be used by designers to provide different levels of abstraction over multi-agent systems. The research in these fields has lead to the formulation of a new version of the SODA methodology where environment abstractions and layering principles are exploited for en- gineering multi-agent systems.

Study of silicon-on-insulator multiple-gate MOS structures including band-gap engineering and self heating effects

The progresses of electron devices integration have proceeded for more than 40 years following the well–known Moore’s law, which states that the transistors density on chip doubles every 24 months. This trend has been possible due to the downsizing of the MOSFET dimensions (scaling); however, new issues and new challenges are arising, and the conventional ”bulk” architecture is becoming inadequate in order to face them. In order to overcome the limitations related to conventional structures, the researchers community is preparing different solutions, that need to be assessed. Possible solutions currently under scrutiny are represented by: • devices incorporating materials with properties different from those of silicon, for the channel and the source/drain regions; • new architectures as Silicon–On–Insulator (SOI) transistors: the body thickness of Ultra-Thin-Body SOI devices is a new design parameter, and it permits to keep under control Short–Channel–Effects without adopting high doping level in the channel. Among the solutions proposed in order to overcome the difficulties related to scaling, we can highlight heterojunctions at the channel edge, obtained by adopting for the source/drain regions materials with band–gap different from that of the channel material. This solution allows to increase the injection velocity of the particles travelling from the source into the channel, and therefore increase the performance of the transistor in terms of provided drain current. The first part of this thesis work addresses the use of heterojunctions in SOI transistors: chapter 3 outlines the basics of the heterojunctions theory and the adoption of such approach in older technologies as the heterojunction–bipolar–transistors; moreover the modifications introduced in the Monte Carlo code in order to simulate conduction band discontinuities are described, and the simulations performed on unidimensional simplified structures in order to validate them as well. Chapter 4 presents the results obtained from the Monte Carlo simulations performed on double–gate SOI transistors featuring conduction band offsets between the source and drain regions and the channel. In particular, attention has been focused on the drain current and to internal quantities as inversion charge, potential energy and carrier velocities. Both graded and abrupt discontinuities have been considered. The scaling of devices dimensions and the adoption of innovative architectures have consequences on the power dissipation as well. In SOI technologies the channel is thermally insulated from the underlying substrate by a SiO2 buried–oxide layer; this SiO2 layer features a thermal conductivity that is two orders of magnitude lower than the silicon one, and it impedes the dissipation of the heat generated in the active region. Moreover, the thermal conductivity of thin semiconductor films is much lower than that of silicon bulk, due to phonon confinement and boundary scattering. All these aspects cause severe self–heating effects, that detrimentally impact the carrier mobility and therefore the saturation drive current for high–performance transistors; as a consequence, thermal device design is becoming a fundamental part of integrated circuit engineering. The second part of this thesis discusses the problem of self–heating in SOI transistors. Chapter 5 describes the causes of heat generation and dissipation in SOI devices, and it provides a brief overview on the methods that have been proposed in order to model these phenomena. In order to understand how this problem impacts the performance of different SOI architectures, three–dimensional electro–thermal simulations have been applied to the analysis of SHE in planar single and double–gate SOI transistors as well as FinFET, featuring the same isothermal electrical characteristics. In chapter 6 the same simulation approach is extensively employed to study the impact of SHE on the performance of a FinFET representative of the high–performance transistor of the 45 nm technology node. Its effects on the ON–current, the maximum temperatures reached inside the device and the thermal resistance associated to the device itself, as well as the dependence of SHE on the main geometrical parameters have been analyzed. Furthermore, the consequences on self–heating of technological solutions such as raised S/D extensions regions or reduction of fin height are explored as well. Finally, conclusions are drawn in chapter 7.

Analysis of optimal control problems for the incompressible MHD equations and implementation in a finite element multiphysics code

This thesis deals with the study of optimal control problems for the incompressible Magnetohydrodynamics (MHD) equations. Particular attention to these problems arises from several applications in science and engineering, such as fission nuclear reactors with liquid metal coolant and aluminum casting in metallurgy. In such applications it is of great interest to achieve the control on the fluid state variables through the action of the magnetic Lorentz force. In this thesis we investigate a class of boundary optimal control problems, in which the flow is controlled through the boundary conditions of the magnetic field. Due to their complexity, these problems present various challenges in the definition of an adequate solution approach, both from a theoretical and from a computational point of view. In this thesis we propose a new boundary control approach, based on lifting functions of the boundary conditions, which yields both theoretical and numerical advantages. With the introduction of lifting functions, boundary control problems can be formulated as extended distributed problems. We consider a systematic mathematical formulation of these problems in terms of the minimization of a cost functional constrained by the MHD equations. The existence of a solution to the flow equations and to the optimal control problem are shown. The Lagrange multiplier technique is used to derive an optimality system from which candidate solutions for the control problem can be obtained. In order to achieve the numerical solution of this system, a finite element approximation is considered for the discretization together with an appropriate gradient-type algorithm. A finite element object-oriented library has been developed to obtain a parallel and multigrid computational implementation of the optimality system based on a multiphysics approach. Numerical results of two- and three-dimensional computations show that a possible minimum for the control problem can be computed in a robust and accurate manner.

Electrospun Polymeric Scaffolds with Enhanced Biomimetic Properties for Tissue Engineering Applications

This PhD Thesis is focused on the development of fibrous polymeric scaffolds for tissue engineering applications and on the improvement of scaffold biomimetic properties. Scaffolds were fabricated by electrospinning, which allows to obtain scaffolds made of polymeric micro or nanofibers. Biomimetism was enhanced by following two approaches: (1) the use of natural biopolymers, and (2) the modification of the fibers surface chemistry. Gelatin was chosen for its bioactive properties and cellular affinity, however it lacks in mechanical properties. This problem was overcome by adding poly(lactic acid) to the scaffold through co-electrospinning and mechanical properties of the composite constructs were assessed. Gelatin effectively improves cell growth and viability and worth noting, composite scaffolds of gelatin and poly(lactic acid) were more effective than a plain gelatin scaffold. Scaffolds made of pure collagen fibers were fabricated. Modification of collagen triple helix structure in electrospun collagen fibers was studied. Mechanical properties were evaluated before and after crosslinking. The crosslinking procedure was developed and optimized by using - for the first time on electrospun collagen fibers - the crosslinking reactant 1,4-butanediol diglycidyl ether, with good results in terms of fibers stabilization. Cell culture experiments showed good results in term of cell adhesion and morphology. The fiber surface chemistry of electrospun poly(lactic acid) scaffold was modified by plasma treatment. Plasma did not affect thermal and mechanical properties of the scaffold, while it greatly increased its hydrophilicity by the introduction of carboxyl groups at the fiber surface. This fiber functionalization enhanced the fibroblast cell viability and spreading. Surface modifications by chemical reactions were conducted on electrospun scaffolds made of a polysophorolipid. The aim was to introduce a biomolecule at the fiber surface. By developing a series of chemical reactions, one oligopeptide every three repeating units of polysophorolipid was grafted at the surface of electrospun fibers.

Controlled reproduction in Anguilla anguilla (L.): advanced studies on broodstock management, spawning techniques and system design for artificial seed production.

The thesis aims to expose the advances achieved in the practices of captive breeding of the European eel (Anguilla anguilla). Aspects investigated concern both approaches livestock (breeding selection, response to hormonal stimulation, reproductive performance, incubation of eggs) and physiological aspects (endocrine plasma profiles of players), as well as engineering aspects. Studies conducted on various populations of wild eel have shown that the main determining factor in the selection of wild females destined to captive breeding must be the Silver Index which may determine the stage of pubertal development. The hormonal induction protocol adopted, with increasing doses of carp pituitary extract, it has proven useful to ovarian development, with a synchronization effect that is positively reflected on egg production. The studies on the effects of photoperiod show how the condition of total darkness can positively influence practices of reproductions in captivity. The effects of photoperiod were also investigated at the physiological level, observing the plasma levels of steroids ( E2, T) and thyroid hormones (T3 and T4) and the expression in the liver of vitellogenin (vtg1 and vtg2) and estradiol membrane receptor (ESR1). From the comparison between spontaneous deposition and insemination techniques through the stripping is inferred as the first ports to a better qualitative and quantitative yield in the production of eggs capable of being fertilized, also the presence of a percentage of oocytes completely transparent can be used to obtain eggs at a good rate of fertility. Finally, the design and implementation of a system for recirculating aquaculture suited to meet the needs of species-specific eel showed how to improve the reproductive results, it would be preferable to adopt low-flow and low density incubation.