Développement des échafaudages à trois dimensions pour la formation directionnelle de réseau d'angiogénèse
The objective of this study is to investigate the possibility to produce surface modified polymeric fibres with the capability of both directional endothelial cell (EC) patterning and inducing angiogenesis in a 3D cell culture system. This study was conducted in three steps as following: 1) surface modification and characterization of materials, and more specifically, polymeric fibre surfaces, involving a multilayer, surface modification approach, using plasma polymer deposition methods, dextran and certain bioactive compounds grafting, to induce predictable biological responses. 2) In vitro evaluation of the surface modified polymer fibres towards EC behaviour in a 2D cell culture system. 3) In vitro evaluation of surface modified, polymer fibres towards EC behaviour (angiogenesis) in three 3D cell culture systems. Initially, the surfaces of 100-?m diameter poly (ethylene terephtalate) (PET) or polytetrafluoroethylene (PTFE) fibres (as monofilaments) were coated, utilising a multilayered-surface modification strategy, performed in three steps. These substrates were initially coated, using the radiofrequency glow discharge deposition technique, with a thin, polymeric interfacial bonding layer, produced via n-hepthylamine plasma polymer (HApp) or an acetaldehyde plasma polymer (AApp), having amine and aldehyde groups, respectively. Carboxy-methyl-dextran (CMD) was then covalently immobilized, using water-soluble carbodiimide chemistry (EDC/NHS), onto the surface amine groups, present either on the HApp-coated or on AApp-PEI-coated substrate surfaces. In the last step, GRGDS peptides were covalently immobilized onto the CMD-coated fibre surfaces. The second research theme involves the in vitro evaluation of surface modified substrates towards EC behaviour. For this purpose, human umbilical vein ECs (HUVECs) were seeded and grown on surfaces to evaluate the cell responses such as cell adhesion, spreading, cytoskeleton reorganization and cell orientation. Some alternative substrates were also examined in order to further characterize the cell behaviour. The cell behaviour was related with the surface physicochemical properties of the test modified surfaces. On CMD-coated substrates, cell adhesion was reduced, in contrast the amine-, aldehyde- and GRGDS-coated substrates promoted cell attachment and spreading and actin filaments and focal adhesions formations. Conversely, the reduced cell adhesion on GRGES (negative control), demonstrates that the increased EC adhesion, on GRGDS-grafted surfaces were attributed to specific biological responses of cell surface integrins towards the RGD ligands present on the surfaces. Cell adhesion was enhanced as the GRGDS solution concentration was increased from 0.1 mg/ml to 1 mg/ml. In comparison with"flat" substrates, fibre curvature promoted cell orientation along the fibre axis. In the third step, surface modified PET polymer fibres were evaluated towards"angiogenesis" in in vitro 3D tissue construct models, using three different methods of cell seeding. The result showed that angiogenesis process occurred when either HUVEC pre-coated fibres were embedded in fibrin gel, or HUVECs and cell-free fibres were sandwiched together between two layers of fibrin gel. These results suggest that the physical effect of the fibres, in conjunction with the surface chemistry, promotes in vitro EC attachment, induces angiogenesis and enhances directional angiogenic structures formation in the fibrin-based models. By prolonging the incubation period, the number of angiogenic structures increased and a network was formed, in which angiogenic structures interconnected to each other, from one fibre to another, following an optimal fibre spacing ranging from 200 to 600 [mu]m. These results demonstrate that through the use of a fibrous polymeric material that is surface coated with cell adhesive materials, in particular with extracellular matrix components such as RGD peptide or gelatin, it becomes possible to both enhance and direct the angiogenic process. Therefore, the two main goals of this study which were (i) to establish the feasibility of pre-vascularizing an in vitro tissue construct, and (ii) to influence the guidance of microvessel growth in a pre-determined direction, using phenomena known as"contact guidance" by means of polymer fibers and as"signaling molecules" by means of ligand-integrin interactions were largely achieved."--Résumé abrégé par UMI.
- Génie – Thèses