DESIGN, SYNTHESIS, CHARACTERISATION AND BIOLOGICAL TESTING OF NEW DERIVATIVES OF HYALURONAN FOR THE PRECONDITIONING OF STEM CELLS FOR THE THERAPY OF MUSCULAR DYSTROPHIES AND THE DEVELOPMENT AND REGENERATION OF SKELETAL MUSCLE

Regenerative medicine is the process of creating living, functional tissues to repair or replace tissue or organ functions lost due to age, disease, damage, or congenital defects. Related to regenerative medicine is field of tissue engineering. Numerous strategies currently used to engineer tissues depend on employing a specific material. These scaffolds serve as a synthetic extra cellular matrix thus to direct the growth and the formation of a desired tissue. Recent work has suggested application of mesoangioblasts for stem cell therapies for muscular dystrophy. Experiments in dystrophic mice and dogs have shown that mesoangioblasts transplantation can restore muscle function. Anyway, the possibility of using this novel strategy for the cell therapy in genetic diseases is strictly related to the capacity to expand mesoangioblasts in culture in a reproducible and efficient way. Hence, the aim of this project is to investigate the ability of the hyaluronic acid (HA) to act as a basement for cells culture; cells are in fact very sensitive to their environment and the contact with something mimicking the extra cellular matrix could, in theory, assure a better proliferation and differentiation. Hyaluronan can be chemically modified to introduce drugs, ligands or other bioactive molecules, or to cross link the linear chains affording reticulated networks. Several different methods to provide modified HA have been performed in order to develop robust protocols; specifically we focused on Sharpless cycloaddition, on the use of diisocyanates as cross linker agents and on the activation of the carboxylic acid moiety towards amines. Mesoangioblasts were cultured using HA and its derivatives as substrates to evaluate their biological influence on in vitro proliferation and differentiation. Furthermore a deep investigation concerning the kinetics of the Michael-type addition was performed. Due to its versatility, Michael addition is a precious tool for the bioconjugation often used also for HA-derivatisation. Thus we deeply studied the effect of the reagents architecture (influence of the Michael donor and acceptor), their stability and that of the Michael adducts.