Sacco, Anna Maria (2015) BIOMIMETIC SCAFFOLDS AND CARDIAC FIBROBLAST-DERIVED EXTRACELLLULAR MATRIX FOR STEM CELL-BASED MYOCARDIAL REGENERATION. [Tesi di dottorato]

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Abstract

Recent findings indicate that the number of cardiac primitive cells in the adult human heart increases significantly in pathological conditions. The fact remains, however, that those cells fail to accomplish cardiac tissue regeneration in chronic pathological conditions in vivo. Similarily, therapeutic stem cell delivery, either intravenous, intracoronary or intramyocardial, and activation, for instance by genetic modification or local growth factor injection, have yielded moderate and controversial results that make stem cell-based myocardial regeneration still merely an experimental approach to cardiac disease treatment. Adverse effects of underlying pathology, with cellular senescence and microenvironment modifications, might be responsible for such outcome. The aims of the present research were to characterize cardiac primitive cells in the normal and pathological (ischemic cardiopathy) adult human heart, to produce and characterize cardiac fibroblast-derived extracellular matrix in vitro, and to characterize cardiac primitive cells cultured in the presence of this substrate in terms of their survival, proliferation, migration, and maturation. These findings should then lead to the identification of the biochemical signalling molecules, i.e. bioactive components of cardiac extracellular matrix, to be integrated in the scaffolding bioartificial materials and allow the evaluation of the bioactivity of scaffolds incorporating extracellular matrix proteins. Thus, the final aim of the study was to develop bioactive scaffolds populated with cardiac primitive cells for the regeneration of infarcted myocardial tissue, based on bioactive and tissue-specific molecules, exerting the same biochemical signals of the natural extracellular matrix during heart development and regeneration. Cardiac primitive cells and cardiac fibroblasts were isolated from samples of cardiac tissue derived from donor hearts and from hearts explanted due to ischemic cardiopathy in patients with end-stage heart failure. The population of cardiac primitive cells and the extracellular matrix deposited by cardiac fibroblasts in vitro were characterized by immunohistological methods, immunoblotting, immunoabsorbent enzymatic assay, or RT-PCR. Cardiac primitive cells from normal and pathological hearts were cultured in the presence of normal and pathological cardiac fibroblast-derived matrix, cardiac fibroblast-conditioned medium, or polyurethane scaffolds functionalized with gelatin or laminin-1 and their proliferation, apoptosis, migration and maturation were evaluated by BrdU incorporation assay, TdT assay, scratch wound assay and RT-PCR, respectively. The results of the study highlighted the role of microenvironment in cardiac regeneration. Changes that take place in chronic pathological conditions should be taken into consideration when planning stem cell-based therapy. Cardiac fibroblast-derived matrix can be used for in vitro studies of the interactions between components of extracellular matrix and cardiac primitive cells responsible for cardiac self-renewal in normal and pathological conditions. The goal of regenerative medicine and tissue engineering to regenerate damaged myocardium can be achieved by further investigations into cell-matrix interactions at different stages of cardiac ischemic disease progression.

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