Pedram, Parisa (2021) Engineering novel micro-scaffolds and bottom up strategies for in vitro building of vascularized hybrid tissues. [Tesi di dottorato]

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There is a significant demand of de-novo engineered tissue grafts capable of replacing biological tissue and/or organ functions for clinical applications. The major obstacle to achieve this goal is the difficulty of recreating all of the complex biochemical and biomechanical functions of the tissue to regenerate, together with transport limitations into the bulk of these newly synthesized tissues. Oxygen and nutrients are supplied to cells and tissues naturally by the microvasculature, which is composed of branching, variable diameter blood vessels. Replicating the complex architecture and functionalities of native tissue vasculature is therefore one of the most important challenge in tissue engineering strategies. To date, bottom up techniques are strong powerful tools to build large viable tissue constructs by packing and sintering cell-laden scaffold-based micro-modules (μ-scaffolds) in a mould. In fact, after sintering and further μ-scaffolds degradation it is possible to achieve large viable tissues in vitro, replicating the composition and structure of native tissue and suitable for studying biological processes involved in new tissue genesis, maturation and remodelling. The aim of this work is to design and engineering novel μ-scaffolds and bottom up assembly techniques to fabricate vascularized layered tissues and to study the effect of μ-scaffolds spatial distribution and co-culture of human dermal fibroblasts (HDFs) together with human umbilical vein endothelial cells (HUVECs) on new tissue growth and vascularization in vitro. To achieve these aims, in first part of this study, we fabricated porous polycaprolactone (PCL) μ-scaffolds with bioinspired trabecular structure and we demonstrated that these newly developed μ-scaffolds supported the in vitro adhesion, growth, and biosynthesis of HDFs. The μ-scaffolds were fabricated by using a fluidic emulsion/porogen leaching/particle coagulation process and by using polyethylene oxide (PEO) as a biocompatible pore-generating agent. In particular, the effect of the composition of the polymeric solution and the flow rate of the continuous phase on μ-scaffolds size distribution, morphology and architectural properties were assessed with the aim to find the best preparation conditions for biological characterization. In vitro culture of HDFs showed that μ-scaffolds supported cells adhesion, colonization, proliferation and biosynthesis in the entire three-dimensional porosity up to 25 days. The second part of this study involved the development of a soft-lithography approach to control the spatial assembly of μ-scaffolds and to create two distinctive μ-scaffolds patterns, namely ordered and disordered. The as obtained patterns were used as substrate for culturing HDFs and 11 HUVECs aiming to develop viable monolayers and bilayers tissue constructs in vitro. The results of this study demonstrated that μ-scaffolds patterning directed cells colonization and biosynthesis and guided the morphology and distribution of newly formed vasculature. All of the findings reported in this work demonstrated the vital role of μ-scaffolds architectural features and assembly on in vitro tissue growth and, pay the way about the possibility to create in silico-designed vasculatures inside modularly engineered biohybrids tissues.

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