Controlling the supramolecular organisation of adsorbed collagen layers.

The supramolecular organisation of collagen adsorbed on polymer substrates was investigated as a function of properties of the substrates (chemical nature, roughness) and of characteristics of the collagen solution (concentration, state of aggregation) as well as details of the preparation procedure (adsorption time, drying rate). Elongated structures are formed at the interface by assembly of collagen molecular segments protruding into the solution. This is favoured by using a hydrophobic and smooth substrate, by increasing the adsorbed amount and by increasing the adsorption time, even beyond stages at which the adsorbed amount does no longer vary. Collagen adsorbed at low amount on hydrophobic substrates strongly reorganises into a net-like pattern if drying is performed at low rate. This is due to dewetting and collagen displacement by the water meniscus. Applications derived from the control of collagen organisation are presented. Nanostructured polymer surfaces were created starting from a collagen template. The attachment and the cytoskeletal organisation of mammalian cells (MCF-7/6) were also shown to depend on collagen organisation.

Nanostructured collagen layers obtained by adsorption and drying.

The supramolecular organization of collagen adsorbed from a 7 microg/ml solution on polystyrene was investigated as a function of the adsorption duration (from 1 min to 24 h) and of the drying conditions (fast drying under a nitrogen flow, slow drying in a water-saturated atmosphere). The morphology of the created surfaces was examined by atomic force microscopy (AFM), while complementary information regarding the adsorbed amount and the organization of the adsorbed layers was obtained using radioassays, X-ray photoelectron spectroscopy (XPS), and wetting measurements. The collagen adsorbed amount increased up to an adsorption duration of 5 h and then leveled off at a value of 0.9 microg/cm2. For samples obtained by fast drying, modeling of the N/C ratios obtained by XPS in terms of thickness and surface coverage, in combination with the adsorbed amount, water contact angle measurements and AFM images, indicated that the adsorbed layer formed a felt starting from 30 min of adsorption, the density and/or the thickness of which increased with the adsorption time. Upon slow drying, the collagen layers formed after adsorption times up to about 2 h underwent a strong reorganization. The obtained nanopatterns were attributed to dewetting, the liquid film being ruptured and adsorbed collagen being displaced by the water meniscus. At higher adsorption times, the organization of the collagen layer was similar to that obtained after fast drying, because the onset of dewetting and/or collagen displacement were prevented by the high density of the collagen felt.

Influence of substrate hydrophobicity on the adsorption of an amphiphilic diblock copolymer.

The adsorption of poly(tert-butylmethacrylate)-block-poly(2-(dimethylamino-ethyl) methacrylate) (PtBUMA-b-PDMAEMA) was studied by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) analysis performed on dried samples. The copolymer was dissolved in toluene at concentrations below (0.01 wt%) and above (0.05 and 1 wt%) the CMC; silicon (SiOH) and CH(3)-grafted silicon (SiCH(3)) were used as substrates. Whatever the concentration and the substrate, a layer of individual copolymer molecules, 1.5-3 nm thick, formed rapidly. The adsorbed amount was slightly higher and the resistance to AFM tip scraping was stronger on SiOH than on SiCH(3). This is attributed to hydrogen bonding between the PDMAEMA block and the OH groups of the silicon surface, leading to polarization of the adsorbed layer. Above the CMC, on SiOH, randomly scattered dot-like features (about 5 nm high) observed by AFM were attributed to individual micelles, which were not displaced by drying. On SiCH(3), the particles found on the top of the adsorbed layer were micelle aggregates, about 50 nm thick, the lateral size of which was strongly influenced by the rate of drying. This further difference between SiCH(3) and SiOH is tentatively attributed to the exposure of PDMAEMA by the adsorbed layer formed on SiCH(3), while only PtBUMA would be exposed by the layer adsorbed on SiOH. The red blood cell shape and the size of the micelles observed in single layers indicate that the PtBUMA corona was not made compact as a result of drying.

Radiolabeling of Pluronic amphiphilic copolymer for adsorption studies.

A new pathway for the radiolabeling of Pluronic PE6800 was developed. In a first step, the CH(2)-OH end groups of the copolymer were substituted by tosylates; in a second step these were reduced by [3H]-NaBH(4) to obtain tritiated chain ends. The final product was shown to be a mixture of native, tosylated, and reduced Pluronic containing 1 tritium atom per 1110 Pluronic molecules. The labeling procedure did not affect the molecular weight distribution nor the adsorption isotherm of the copolymer on polystyrene plates. A plateau value of about 0.7 microg/cm(2) is reached at a concentration in solution of 500 microg/ml, i.e., much lower than the cmc. Upon drying, the Pluronic adsorbed layer reorganizes in particles with a size of about 30 to 60 nm which cover about 15% of the substratum surface. This observation is of great importance for the design of protein-resistant surfaces by adsorption of Pluronic.

Surface characterization of poly(methyl methacrylate) microgrooved for contact guidance of mammalian cells.

High-resolution patterns of grooves have been made in poly(methyl methacrylate) films, PMMA, by an electron-beam microlithographic process. The surface of films processed over a large width was characterized in terms of chemical composition (X-ray photoelectron spectroscopy (XPS). time of flight secondary ion mass spectroscopy), wettability (sessile drop) and topography (atomic force microscopy). Collagen adsorption was also studied (radiocounting, XPS) as such or in competition with Pluronic F68. The chemical alteration of the surface induced by the electron-beam irradiation disappeared after the dissolution involved in the development process. W138 human fibroblasts cultivated on microgrooved substrata (grooves 1 microm deep and 0.5-10 microm wide) showed a strong orientation parallel to the grooves. The contact guidance is induced by the topography of the surface and not by the alternation of zones with different physico-chemical properties. It may be explained in terms of probability of successful substratum contact by cell protrusions.