Jean-Benoit Lhoest

Fibronectin adsorption, conformation, and orientation on polystyrene substrates studied by radiolabeling, XPS, and ToF SIMS.

Protein adsorption is widely studied by a variety of techniques, but there still is little known about protein orientation and conformation after adsorption. This probably is due to the large number of parameters involved, such as the characteristics of the surface and the structure of the protein. In this study, the adsorption of fibronectin was investigated with three different techniques: radiolabeling, X-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (ToF SIMS) on polystyrene and oxidized polystyrene. The first two techniques have been widely used to study protein adsorption, allowing us to determine the amount of protein adsorbed on each surface. The ToF SIMS, however, is a technique just emerging for the study of protein adsorption. This study confirms its utility since ToF SIMS is found to be sensitive to the protein orientation and/or conformation at the surface. Indeed, the ToF SIMS peaks characteristic of the protein show differences in their reduced intensity between the two substrates. These differences, which are not detected by XPS, are attributed to different orientations and/or conformations of the protein.

Adhesion of mammalian cells to polymer surfaces: from physical chemistry of surfaces to selective adhesion on defined patterns

The study of the adsorption of type I collagen from a solution containing Pluronic F68 has shown that the latter prevents collagen adsorption on polystyrene and does not prevent it on surface-oxidized polystyrene. This explains the control of mammalian cell adhesion by substrate surface hydrophobicity and composition of pre-conditioning solution. On that basis, selective adhesion of different types of mammalian cells (PC12 pheochromocytoma, MSC80 schwannoma, Hep G2 hepatoblastoma, rat hepatocytes) on patterned surfaces was achieved. Therefore tracks (width in the range of a few tens of microm) of reduced hydrophobicity were produced on polystyrene by photolithography and oxygen plasma treatment. After conditioning by a solution containing both Pluronic F68 and extracellular matrix protein (collagen, fibronectin), the latter adsorbed selectively on these paths thus allowing selective adhesion of the cells.

Orientation of cell adhesion and growth on patterned heterogeneous polystyrene surface

Studies of neurite outgrowth or cell migration, two important processes in neuronal networks formation, are facilitated by cell culture models capable of orientating cellular growth and of designing a well-defined cellular pattern. Heterogeneous polystyrene surfaces composed of oxygen plasma-treated stripes (PSox) with a low hydrophobicity separated by non-treated areas (PS) have these properties. In this study, to guide cell growth, we developed a cell culture model using these supports and we identified the molecular factors involved in cellular orientation. When the heterogeneous supports were not coated, proteins from a serum culture medium were required for cells to line up on PSox. On the other hand, cell orientation on coated surfaces was clearly influenced by competitive adsorption of adhesive proteins such as fibronectin or collagen and anti-adhesive molecules as pluronic F68 or albumin. Attachment factors were adsorbed on PSox stripes while adsorption of anti-adhesive molecules on the most hydrophobic PS areas prevented cell adhesion or growth. Thus, we describe the preparation of a cell culture substrate that succeeded in orientating cell growth and that led to a line of cells on adhesive PSox stripes ranging from 2 to 100 microns width.

Fibronectin-pluronic coadsorption on a polystyrene surface with increasing hydrophobicity: relationship to cell adhesion.

Recently, patterned polystyrene surfaces containing hydrophobic (PS) and more hydrophilic (PSox) areas have been shown to be capable of directing cellular growth, which is mainly due to the competitive adsorption of adhesive and antiadhesive molecules. In this article, the competitive adsorption between a pluronic surfactant and fibronectin was studied on homogeneous PS or PSox substrates conditioned with mixtures containing increasing concentrations of one of the two molecules. Radiolabeling and X-ray photoelectron spectroscopy techniques showed that fibronectin adsorption increased on both surfaces if the fibronectin concentrations increased in the conditioning mixture. In contrast, fibronectin adsorption decreased on PSox and did not occur on PS surfaces when pluronic concentrations increased in the coating mixture. A comparison of these data with pheochromocytoma and Schwann cells cultured on patterned surfaces showed that the direction of cell growth on PSox areas depended first on the relative concentrations of the two components in the mixtures, and second, on their ratio; the best concentration ratio probably depends on the cells ability to recondition its support.

A new plasma-based method to promote cell adhesion on micrometric tracks on polystyrene substrates

A new procedure has been developed in order to obtain heterogeneous polymer surfaces for the promotion of cell adhesion. For this purpose, a microelectronic photosensitive resin was spin coated on polystyrene (PS) substrates. The resin was then submitted to UV light irradiation through a mask and partially developed. The sample was further submitted to a plasma oxygen discharge prior to dissolution of the remaining resin. The characterization by time of flight secondary ion mass spectrometry (ToF SIMS), X-ray photoelectron spectroscopy (XPS), and dynamic contact angle (DCA) allowed us to conclude that hydrophilic paths were created on the more hydrophobic PS substrate together with the complete removal of the resin. In order to optimize cell adhesion contrast, the modified surfaces were then conditioned with a solution containing both a surfactant (pluronic F68) and a protein. Two different proteins were tested (collagen I and fibronectin). PC12 cell cultures on those conditioned surfaces showed that cell adhesion occurs only on the hydrophilic tracks. ToF SIMS spectra and images recorded on those substrates revealed the presence of the proteins only in the hydrophilic tracks. In the same time, the surfactant is suspected to adsorb mainly on the hydrophobic areas of the samples.