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 mu m width. (C) 1998 Elsevier Science B.V. All rights reserved

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

Biological evaluation of RGD peptidomimetics, designed for the covalent derivatization of cell culture substrata, as potential promotors of cellular adhesion.

Our aim was to replace the proteins and peptides, generally used for the biocompatibilization of polymer substrata, with synthetic molecules mimicking the RGD (Arg-Gly-Asp) active sequence. Based on the (L)-tyrosine template, RGD peptidomimetics were constructed; one molecule 3 was equipped with an anchorage arm that allowed its covalent grafting on a culture substratum made from poly(ethylene terephthalate) (PET) microporous membrane. The amount of fixed molecules was readily determined by XPS, using a fluorine tag incorporated in the peptidomimetic structure. The binding of peptidomimetics 1-3 to the vitronectin (VN) and fibronectin (FN) receptors could not be revealed in a test of inhibition of MSC 80 cells adhesion, by the synthetic compounds in solution placed in competition with the adhesive proteins (VN and FN) coating polystyrene plates. However, the cell-attachment activity of peptidomimetic 3 was shown by culturing CaCo2 cells, in the absence of serum, on the PET substratum grafted with 3. The performance of this support was similar to that of PET grafted with the reference peptide RGDS (Arg-Gly-Asp-Ser), and only reduced by half comparatively to the PET grafted with FN

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

Expression of integrins by murine MSC80 Schwann cell line: relationship to cell adhesion and migration.

Schwann cells (Sc) are one of the most important factors promoting regeneration of both the peripheral and the central nervous system. They provide a permissive environment for neurite outgrowth and the making of this environment requires interactions between Sc and extracellular matrix proteins that are mediated via integrin receptors. This study characterized, by immunoprecipitation, the integrins expressed by the mouse MSC80 Sc line. Our results showed that MSC80 Sc expressed alpha1beta1, alpha5beta1 and alpha6beta1 integrins as well as the alpha v-subunit associated with an unidentified 80-90 kDa beta-subunit. Adhesion and migration assays revealed a hierarchy of protein influences that are dependent upon the type of cellular behaviour. Integrin expression correlated with MSC80 Sc line adhesion and migration on extracellular matrix proteins. The MSC80 Sc line expressed a pattern of integrins which allowed adherence on vitronectin and collagen IV, and faster migration on merosin and laminin. As the integrin pattern and the behaviour of MSC80 on ECM were similar to primary Sc, MSC80 are a potential abundant source of Sc for further in vitro and in vivo experiments