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.

The coupling between electron transfer and Protein/Solvent Dynamics in Photosynthetic Reaction Centers: Spectroscopic Studies in Amorphous Matrices

We investigated at the molecular level protein/solvent interactions and their relevance in protein function through the use of amorphous matrices at room temperature. As a model protein, we used the bacterial photosynthetic reaction center (RC) of Rhodobacter sphaeroides, a pigment protein complex which catalyzes the light-induced charge separation initiating the conversion of solar into chemical energy. The thermal fluctuations of the RC and its dielectric conformational relaxation following photoexcitation have been probed by analyzing the recombination kinetics of the primary charge-separated (P+QA-) state, using time resolved optical and EPR spectroscopies. We have shown that the RC dynamics coupled to this electron transfer process can be progressively inhibited at room temperature by decreasing the water content of RC films or of RC-trehalose glassy matrices. Extensive dehydration of the amorphous matrices inhibits RC relaxation and interconversion among conformational substates to an extent comparable to that attained at cryogenic temperatures in water-glycerol samples. An isopiestic method has been developed to finely tune the hydration level of the system. We have combined FTIR spectral analysis of the combination and association bands of residual water with differential light-minus-dark FTIR and high-field EPR spectroscopy to gain information on thermodynamics of water sorption, and on structure/dynamics of the residual water molecules, of protein residues and of RC cofactors. The following main conclusions were reached: (i) the RC dynamics is slaved to that of the hydration shell; (ii) in dehydrated trehalose glasses inhibition of protein dynamics is most likely mediated by residual water molecules simultaneously bound to protein residues and sugar molecules at the protein-matrix interface; (iii) the local environment of cofactors is not involved in the conformational dynamics which stabilizes the P+QA-; (iv) this conformational relaxation appears to be rather delocalized over several aminoacidic residues as well as water molecules weakly hydrogen-bonded to the RC.