Immobilisation orientee du facteur de croissance de l'epiderme a des fins d'application en ingenierie tissulaire

In this work, we have captured a coil-tagged growth factor, EGF, in an oriented fashion through its interaction with a surface on which the coil partner will be covalently immobilized. We selected EGF since this growth factor is known to have a major impact on cellular proliferation during wound healing mechanisms, which make it an excellent candidate for tissue engineering applications.;In a second paper, we grafted Ecoil-EGF on aminated glass surfaces. The efficiency of our grafting procedure that was based on the covalent immobilization of K coil using a heterobifunctional linker, followed by Ecoil-EGF capture via coiled-coil interactions, was tested with a biosensor whose detection principle is based on surface plasmon resonance (SPR). This study confirmed the high specificity and stability of the coiled-coil interactions. Each grafting step was further evaluated by ellipsometric and contact angle measurements on glass surfaces. An increase of the thickness of the surface and a modification of its hydrophobicity were observed after each grafting step. These results confirmed the covalent grafting of K coil peptides and the oriented immobilization of Ecoil-EGF (115 +/- 8 pmol/cm2) on aminated glass surfaces. A-431 cells were finally incubated on native surfaces (in the presence or absence of soluble EGF), on Kcoil-functionalized surfaces on which Ecoil-EGF was captured via coiled-coil interactions or on which EGF was covalently immobilized. A higher cellular response (cell adhesion and receptor phosphorylation) was observed for coiled-coil immobilized Ecoil-EGF when compared to the other approaches we have tested.;At last, we evaluated the impact of Ecoil-EGF tethering on polyethylene terephthalate (PET) via coiled-coil interactions upon human corneal epithelial cell culture (HCE-2). First, amine groups were created on PET surface by ammonia plasma treatment. K coil peptides were covalently immobilized using the same protocol as the one developed for aminated glass and the grafting process was characterized by X-Ray Photoelectron Spectroscopy (XPS) analyses. As expected, K coil peptides were covalently grafted, however, a surface treatment with human serum albumin (HSA) was required to avoid non-specific adsorption of EGF. The impact of oriented immobilized EGF (46.9 +/- 9.5 pmol/cm 2) on HCE-2 behavior was then studied. First, an increase of HCE-2 adhesion was observed in the presence of Ecoil-EGF immobilized via coiled-coil interactions, in stark contrast with results obtained with PET (with or without soluble EGF supply in solution) or in the presence of PET on which EGF had been physically adsorbed. 24h after inoculation, cell spreading was also enhanced in the presence of immobilized Ecoil-EGF whereas cells remain spherical with other conditions. Moreover, proliferation assays demonstrated that immobilized Ecoil-EGF was the most efficient condition to promote cell growth, most likely due to the prolonged activation of their EGF-triggered signaling pathways. (Abstract shortened by UMI.);In our strategy, EGF was first expressed as a fusion protein comprising the E or the K coil at its N-terminus. Other EGF fusion proteins were also expressed for comparison. These included EGF being tagged with the Fc portion of an Immunoglobulin G at its N- or C-terminus, as these constructs have been previously reported for the oriented immobilization of EGF via their interaction with protein G. In a first step, we have studied the impact of the nature and the localization of different tags on EGF bioactivity, which is the subject of a first manuscript that was published in 2008 in Tissue Engineering part A. The various EGF constructs were expressed by transiently transfecting human embryonic kidney (HEK 293) cells and they were purified by affinity chromatography. The bioactivity of these proteins was then evaluated. It was demonstrated that purified Fc-EGF was not able to promote EGFR phosphorylation whereas Ecoil-EGF was as active as untagged EGF and more active than the others proteins including EGF-Fc and Kcoil-EGF. These results demonstrated that, in our approach, the Ecoil peptide is the only tag that does not interfere with EGF bioactivity and that the nature and the localization of the tag have a strong influence on EGF chimera bioactivity. We have thus decided to use Ecoil-EGF in all subsequent work.