Jean-Luc Dewez

Competitive adsorption of proteins: key of the relationship between substratum surface properties and adhesion of epithelial cells.

The adhesion of Hep G2 cells was investigated using different substrata (commercial substrata, polystyrene modified by oxygen or ammonia plasma discharge), the surface properties of which were characterized (surface chemical composition, water contact angle, zeta potential). Some substrata were pre-conditioned with solutions of extracellular matrix (ECM) protein (collagen, laminin, fibronectin), solutions of albumin or polylysin, fetal calf serum or culture medium. The culture medium contained the surfactant Pluronic F68; cycloheximide was added in certain tests to inhibit protein synthesis. Cells spread within 1.5 h provided ECM proteins were present at the surface. Adsorption of ECM proteins was subject to competition with adsorption of Pluronic F68. When the substratum was exposed simultaneously to ECM protein and Pluronic F68, either by pre-conditioning or through protein cell secretion, a weaker substratum hydrophobicity favored adsorption of the proteins and subsequent cell adhesion. On the other hand, when ECM proteins were pre-adsorbed, they were not displaced by Pluronic F68 and cell adhesion was not influenced by substratum hydrophobicity. When ECM proteins were present, no difference was observed between substrata of similar hydrophobicity carrying positive or negative charges, respectively. In absence of ECM proteins, the presence of cationic sites at the substratum surface (NH3 plasma treatment, adsorption of polylysine) allowed cell attachment but no spreading within 1.5 h.

Surface hydroxylation of poly(vinylidene fluoride) (PVDF) film

The surface of PVDF film was selectively modified by wet chemistry. Treatment with aqueous LiOH produced HF-elimination and the emergence of an oxygen-containing functionality. The XPS analysis clearly indicated the presence of ketone-, ether(epoxide)-, and alcohol motifs. The percentage of alcohols could be significantly increased by reduction of the ketones with NaBH4 in 2-propanol, followed by reduction of the epoxides with DIBAL-H in hexane. Thus, the full treatment led to a PVDF surface displaying 7 to 16% of oxygen-containing units, of which about 60% consisted in alcohol motifs. The reactvity of the surface-displayed hydroxyl functions was assayed by radiolabeling with [H-3]-Ac2O

Coupled influence of substratum hydrophilicity and surfactant on epithelial cell adhesion.

The influence of substratum surface hydrophilicity and of a surfactant on human epithelial cell adhesion and protein adsorption was investigated. Therefore, tissue culture grade polystyrene (TCPS) and bacteriological grade polystyrene (BGPS) substrata were treated with different media, with or without Pluronic F68 [a poly(ethylene oxide) and poly(propylene oxide) triblock copolymer surfactant], and with or without type I collagen as a typical extracellular matrix protein. The conditioned substrata were submitted to XPS analysis and assayed for cell adhesion by inoculating Hep G2 cells in a chemically defined nutritive medium. The presence of collagen at the substratum surface is required to obtain attachment and spreading of Hep G2 cells. With PS substrata, treating with a solution of collagen does not promote cell adhesion if the solution contains Pluronic; XPS data show that this is due either to prevention of collagen adsorption or to its desorption by rinsing. With less hydrophobic TCPS substrata, the presence of Pluronic in the conditioning solution does not preclude cell adhesion, nor collagen adsorption. The effect of BGPS and TCPS substrata on Hep G2 cell adhesion is thus mediated by the presence of a surfactant that affects the adsorption of collagen.

Reactivity assay of surface carboxyl chain-ends of poly(ethylene terephthalate) (PET) film and track-etched microporous membranes using fluorine labelled- and/or 3H-labelled derivatization reagents: tandem analysis by X-ray photoelectron spectroscopy (XPS) and liquid scintillation counting (LSC)

Poly(ethylene terephthalate) (PET) films and track-etched microporous membranes of two different porosities were pretreated by hydrolysis and/or oxidation in order to enhance the amount of carboxyl chain-ends displayed on their surface. The reactivity of these carboxyl functions was determined by derivatization assays in which the reactions were carried out under conditions likely to be encountered in the coupling of water-soluble biochemical signals on the surface of biomaterials. Original reagents, fluorine-labelled and/or H-3-labelled aminoacid compounds, were used. The derivatized PET samples were examined by X-ray photoelectron spectroscopy (XPS) to characterize their apparent surfaces, and by liquid scintillation counting (LSC) to quantify the amount of tags fixed on their open surfaces. Using this dual assay technique, we analyzed the surface of microporous membranes which are currently used as substrates for cell culture systems.