The control of morphology is of particular relevance in fields as diverse as adhesion, wetting, tribology, optics, biomaterial design, etc.

Different methods have been developed, allowing the creation of surface topography at the micrometer scale. These include particle blasting (Wennerberg et al, 1995), laser ablation (Hopp et al, 1996), settling of microspheres (Miyaki et al, 1999), methods based on photolithography (Torimitsu and Kawana, 1990 ; Ito et al, 1997; Jackman et al, 1999; Walboomers et al, 1999) or on microcontact printing (Kumar and Whitesides, 1994). More recently, the interest has also been focused on the production of surface patterns at the nanometer scale, using phase separation (Imabayashi et al, 1998) or nanolithography (Xu and Liu, 1997) in self-assembled monolayers, electron beam lithography (Hiroshima and Komuro, 1993), imprint lithography (Chou et al, 1996), or colloidal lithography (Burmeister et al, 1999). These topographical changes at the micro-or the nanometer scale are, in most cases and to a lesser or a greater extent, accompanied by chemical modifications of the surface. Curtis and Wilkinson (1999) recently reviewed the reactions of mammalian cells to nanotopography. They concluded that many questions remained unanswered and, in that view, the design of new nanostructured surfaces with controlled chemistry is an important challenge.

Crystalline protein monolayers originating from the surface of bacteria, so-called S-layers, have been used to control the etching of a thin metal coating, producing a periodic array of 10-nanometer holes, in which the underlying graphite substrate was exposed (Douglas et al, 1992). The surface organization of adsorbed proteins varies according to the physico- chemical properties of the substrate and the adsorption parameters, thus opening the way to a large variety of nanostructures. Collagen adsorbed on mica was shown to form large fibrils at high concentration and pH, and a fine meshwork at low concentration and pH (Chernoff and Chernoff, 1992). Collagen presented different nanoscale organizations on different polymers (Dufrene et al, 1999b); this was also observed for a mussel adhesive protein (Baty et al, 1997). Changes in substrate surface properties combined with drying were shown to affect the surface organization as well, with the production of particular patterns in some

cases (Mertig et al, 1997; Dufrne et al, 1999a; Baty et al, 1996; Lestelius et al, 1997; Emch et al, 1991). Controlling the rate of drying after adsorption of collagen on poly (methyl methacrylate) (PMMA) allowed varying the surface organization, from a continuous to a discontinuous adsorbed layer at high and low drying rates, respectively (Dupont-Gillain et al, 1999).

Spin-coating with polymer solutions is commonly used to planarize surfaces, i. e. to reduce or eliminate topographic features. Many theoretical and experimental studies have led to the determination of a range of parameters favoring planarization. The most important are: high film thickness, low viscosity solution, low molecular weight polymer, low volatility solvent, low spinning speed. The geometry and the location of the surface asperities to be planarized are determinant as well (Stillwagon and Larson, 1988 and 1990; Gu et al, 1995; Bullwinkel et al, 1995; Schiltz, 1995; Gupta and Gupta, 1998; Wu and Chou, 1999).

In a first aspect the invention provides a method for controlling the morphology of the surface of a polymer comprising the steps of: depositing on said surface a template layer of a substance which is distributed according to any suitable design acting as a template for a final surface structure; and applying on said template layer a solvent corresponding to said polymer or a mixture of said solvent and the polymer.

A preferred embodiment includes a template deposition which is performed via adhesion, fastening, chemical binding, adsorption, solvent evaporation or spin-coating.

A yet more preferred embodiment includes a drying step which is performed once the template layer is deposited on the surface.

Preferred polymers are chosen from the group comprising of polyamides, polyoleofins, polyesters, polyacrylates such as PMMA and PMA, polystyrene or elastomers. Solvents corresponding to these polymers are known in the art. The deposition of the template layer is preferably obtained by spin-coating.

In a second aspect the invention relates to the polymer surface obtainable by the method according to the invention. Several advantageous uses will be disclosed.

The invention will be disclosed in detail in combination with the drawings wherein the result of several embodiments of the method according to the invention are elucidated. In the drawing: Figure 1 shows atomic force microscope images (5 x 5 lum2) of (a) a poly (methyl methacrylate) substrate (vertical grey scale = 10 nm); (b) the same after formation of a template by adsorption of collagen, rinsing and drying (vertical grey scale = 10 nm); (c) the same as b after spin-coating with chlorobenzene (vertical grey scale =100 nm).

Figure 2 shows atomic force microscope images (a: 2 x 2 lum2 and vertical grey scale = 7 nm; b, c: 5 x 5 Nm2 and vertical grey scale = 10 nm; d: 5 x 5 um2 and vertical grey scale = 20 nm) of the conditioning layer obtained by changing details of the procedure: (a) PMMA + conditioning layer applied using high or low drying speed; (b) use of surface-oxidized and non modified polystyrene as substratum; (c) use of different concentrations of collagen for adsorption on polystyrene; (d) use of different water film thicknesses when the drying step is initiated. In a top, b top and c bottom, the conditioning layer is continuous, which is not suitable as template. In the other cases, the template design varies from a film pearced with holes, to a net, to a broken net.

Figure 3 shows AFM images (size = 5 x 5 Nm2 ; z-range = 10 nm) of PMMA substrate after PBS conditioning (a), and subsequent spin-coating with pure chlorobenzene (b) or with a 2.0 g/i PMMA solution (c).

Figure 4 shows AFM images (size = 5 x 5 Nm2) of samples prepared by collagen adsorption and fast drying (a; z-range = 10 nm), and of the same after spin-coating with a PMMA solution at concentration of 0.0 (b; z-range = 50 nm), 0.2 (c; z-range = 50 nm), and 2.0 g/I (d; z-range = 100 nm). A cross section taken along the horizontal line indicated by the arrow is shown beneath each image.

Figure 5 shows AFM images (size = 5 x 5, um2) of samples prepared by collagen adsorption and slow drying (a; z-range = 10 nm), and of the same after spin-coating with a PMMA solution at concentration of 0.0 (b; z-range = 100 nm), 0.2 (c; z-range = 100 nm), and 2.0 g/t (d; z-range = 100 nm). A cross section taken along the horizontal line indicated by the arrow is shown beneath each image.

Figure 6 shows the variation of the Rrms as a function of the wavelength for the PMMA substrate after PBS conditioning (O), the PMMA substrate after collagen adsorption (a), the same after spin-coating with chlorobenzene (A), or with a 2.0 gui PMMA solution (s). (a) fast drying after collagen adsorption, the confidence intervals obtained from different sets of data being rather large, the data were extracted from the images presented in Figure 2, for the sake of clarity ; (b) slow drying after collagen adsorption, the error bars show the confidence interval at 95 % percent (at least two sets of data containing at least three images).

Figure 7 shows a schematic representation of a PMMA sample during collagen adsorption.

The parts of the sample intended for AFM imaging and for wetting measurements are indicated.

Figure 8 shows (a) AFM images (5 x 5 Nm2 ; z = 20 nm), and (b) variation of cosO as a function of the three-phases contact line position X during three successive wetting cycles (1,2,3), for PMMA conditioned by collagen and fast dried.

Figure 9 shows AFM images (5 x 5 m2 ; z = 20 nm) of PMMA conditioned with collagen and slowly dried in horizontal position (a) or in vertical position (b, top; c, middle ; d, bottom of the sample). A cross section taken along the line indicated by the arrow is shown beneath each image. (e) Variation of cos6 as a function of the three-phases contact line position X during three successive wetting cycles in water (1,2,3) for the sample characterized by images b, c and d.

Figure 10 shows AFM images (5x51um2) of a PMMA substratum after spin-coating with chlorobenzene or with dioxane (left), of a PMMA substratum after collagen adsorption and slow drying (centre), and of the latter after subsequent spin-coating with chlorobenzene or dioxane (right). A cross-section taken along a horizontal line is shown beneath each image.

Figure 11 shows AFM images (5x5 um2) of a PMMA substratum after spin-coating with dioxane (left), of a PMMA substratum after collagen adsorption and slow drying (centre), and of the latter after subsequent spin-coating with dioxane (right). A cross-section taken along a horizontal line is shown beneath each image.

Figure 12 shows AFM images (5x5 um2) of a PMMA substratum after collagen adsorption and slow drying (left), and after the same treatment followed by spin-coating with dioxane (right). A cross section taken along a horizontal line is shown beneath each image.

Figure 13 shows AFM images (5x5 um2) of a PS substratum after spin-coating with carbon disulfide or dioxane (left), of a PS substratum after collagen adsorption and slow drying (centre), and of the latter after subsequent spin-coating with carbon disulfide or dioxane (right). A cross-section taken along a horizontal line is shown beneath each image.

Figure 14 shows AFM images (5x5 um2) of a PS substratum after spin-coating with toluene (left), of a PS substratum after collagen adsorption and slow drying (centre), and of the latter after subsequent spin-coating with toluene (right). A cross-section along a horizontal line is shown beneath each image.

The invention is directed to a surface treatment for a polymer resulting in a relief showing defined characteristics of morphology : nature (positive or negative relief features), shape (spots, lines, network) and size (from 3 nanometer to above 1 micrometer). In particular it allows a nanostructured surface finish to be obtained with a defined design, imposed by the template. An example of the obtained results is illustrated in figure 1. Figure 1 presents atomic force microscopy (AFM) images illustrating the surface relief typical of each stage of the process according to the invention.

The polymer surface to be treated or substratum may be any organic polymer for which a suitable solvent can be found. In a typical application, the polymer and solvent may be poly (methyl metacrylate) (PMMA) and chlorobenzene, chloroform or dioxane, respectively, or polystyrene and toluene, dioxane or carbon disulfide respectively.

The substratum may be a solid body of any shape. In a typical application it may be a film or a plate with an essentially flat surface. It may be found advantageous to chemically modify this surface, for instance to change its wetting properties.

The layer deposited on the substratum to form a template may be all kind of molecules or particles. In a preferred application, these substances may be brought to the surface via an aqueous solution or a suspension and the required design may be obtained by drying the

layer. The desired design may be obtained by using the adequate protein concentration and by selecting the appropriate drying conditions (sample position, gas flow rate, temperature, humidity, etc.).

A yet more preferred embodiment includes the adsorption of proteins from a solution. The required design may be obtained by using the adequate solvent, protein concentration, adsorption conditions (temperature, time), and by selecting the appropriate rinsing and drying conditions. According to this application the protein may be collagen and the template may have different designs, from a film pierced by holes to a net. Other proteins having a globular shape (albumin, fibrinogen,...), a fibrilla shape (fibronectin...) and a surfactant character offer specific advantages. The diversity of template layer which may be obtained depending on details of the procedure is illustrated by Figure 2.

In another preferred application, the substances used to create the template may be colloidal particles adhering, fastened, chemically bound or adsorbed to the polymer substratum.

Typical examples are oxides (aluminum, iron, titanium, silicon oxides...), clay minerals, polymer colloids (latex,...), metal colloids (gold, platinum, silver...), carbon colloids (fullerenes, nanotubes...). The desired design may be obtained by selecting colloids with the suited surface hydrophobicity, electrical properties or macromolecular coverage, and by choosing the suspension medium (organic solvent or water; pH, ionic strength...).

The solvent treatment used to develop the surface relief according to a design imposed by the template may be performed in several ways. According to the known art the solvent may be selected by considering its volatility and the polymer solubility. For instance the solvents may be selected for the corresponding polymer (Fuchs, 1989). Typical combinations are: - polystyrene and toluene, cyclohexane, carbon disulfide, 1,4-dioxane, 1-nitropropane or a combination thereof, - PMMA and chloroform, methyl ethylketone, chlorobenzene, 1,4-dioxane or a combination thereof, - polypropylene and diethyl ether, heptane, octane, dichloroethylene or a combination thereof, -polyvinylchloride and acetone/carbon disulfide, cyclohexanone, nitrobenzene or a combination thereof.

In a preferred application, the solvent treatment may be applied by a spin-coating procedure.

While the design is imposed by the template, quantitative characteristics (thickness or depth, and width of the relief features) may be controlled by selecting the solvent and adjusting operational factors (rotation speed, acceleration, duration, temperature, partial pressure of the solvent vapor).

Alternative ways are provided by a dip-coating procedure or by a solvent-casting procedure.

Again the process may be optimized by selecting the solvent according to its volatility and to the polymer solubility, and by acting on the rate of solvent evaporation, using experimental factors such as temperature, vapor partial pressure, gas flow. It may be found an advantage to select the solvent according to its surface tension and viscosity, or to dissolve a certain amount of the polymer in the solvent before application.

In the present invention an original strategy to create a modulable polymer surface architecture at the nanometer scale is provided. A preferred embodiment involves adsorption of macromolecules on a polymer surface followed by spin-coating of an appropriate solution.

It takes advantage of the surface organization of the adsorbate to further create a chemically homogeneous but nanometer-scale-tailored polymer surface. Spin-coating is preferably used to selectively achieve planarization, or exacerbate a previously designed surface topography, depending on the selections of substratum, solvent and conditions. The polymer in a preferred embodiment used both as a substrate and for spin-coating is poly (methyl methacrylate) (PMMA), an amorphous polymer widely used to elaborate biomaterials such as contact lenses and teeth and bone cements (Piskin, 1994) and polystyrene, an amorphous polymer used to make cell culture supports (Dewez et al., 1997). The preferred adsorbate is collagen, an extracellular matrix protein. Other examples of polymers and adsorbates are known in the art, and can be chosen by a man skilled in the art.

Several examples and non-limiting embodiments of the invention will be discussed hereunder.

Poly (methyl methacrylate) (PMMA) (Aldrich, Milwaukee, USA; molar mass = 996,000) plates were obtained by compression moulding between two glass plates at 240 °C. For the sake of uniformity, they were all spin-coated (time = 60 s, speed = 5,000 rpm, acceleration = 20,000

rpm/s, temperature = 20 °C) with a 10% (w/w) solution of the same PMMA in chlorobenzene (VEL, Leuven, Belgium).

Collagen (type I from calf skin, Boehringer-Mannheim, Mannheim, Germany) was received as an aqueous solution (3 mg/ml at pH 3.0). It was diluted in phosphate buffered saline solution (PBS) (137 mM NaCI (Merck, Leuven, Belgium), 6.44 mM KH2PO4 (VEL), 2.7 mM KCI (Merck), 8 mM Na2HP04 (VEL)) to a concentration of 7 pg/ml. Adsorption of collagen was performed as follows : a circular PMMA sample (radius = 6 mm) was placed in a well of a tissue culture plate (Falcon, Franklin Lakes, USA); 2 ml of the collagen solution were added and the collagen was allowed to adsorb during 2 h at 37°C. The sample was then rinsed three times, without removal from the solution, by pumping 1. 8 ml of the liquid, adding 1.8 mi of water (HPLC grade produced by a MilliQ plus system from Millipore, Molsheim, France, hereafter referred to as MilliQ) and gently agitating during 5 minutes. After rinsing, the samples were removed from the wells and two different drying procedures were applied. A fast-drying (FD) procedure was performed by flushing the samples with a nitrogen flow ; the samples were then stored in a desiccator containing P205. A slow-drying (SD) procedure was performed by placing the wet samples for a period of 2-3 days in a closed vessel containing a saturated solution of Na2CO3 (VEL), which maintained a relative humidity of about 95%.

Solutions of PMMA in chlorobenzene were prepared at the following concentrations: 0.0,0.2, 0.5,1.0 and 2.0 g/l. 100 pl of these solutions were spin-coated (time = 60 s, speed = 5, 000 rpm, acceleration = 20,000 rpm/s, temperature = 20 °C) on the PMMA samples on which collagen had been previously adsorbed and dried.

In order to evaluate the thickness of PMMA films produced by spin-coating, using the same solutions and parameters with an inert and smooth solid surface, 1 cm2 pieces of silicon wafers were used (Wacker-Chemtronic, Burghausen, Germany).

XPS spectra were recorded using a SSX-100 spectrometer (model 206 from Surface Science Instruments, Mountain View, CA, USA) equipped with a monochromatized aluminum anode (10kV, 12 mA). The angle between the normal to the sample surface and the direction of photoelectron collection was 55°. The order of peak analysis was: survey scan, C1 s, 01 s and N1S, except for the samples prepared with silicon wafers for which the order was : survey scan, Cis, Ois and Si2p. Intensity ratios were converted into molar concentration ratios by

using the sensitivity factors proposed by the manufacturer (Scoffield photoemission cross- sections, variation of the electron mean free path according to the 0.7th power of the kinetic energy, constant transmission function).

AFM images were obtained, in ambient conditions, in the contact mode using a commercial microscope (Nanoscope 111, Digital Instruments, Santa Barbara, CA, USA) equipped with Si3N4 triangular levers (Park Scientific Instruments, Sunnyvale, CA, USA; typical radius of curvature = 20 nm, typical spring constant = 0.01 or 0.03 N/m). The scan rate was 1 Hz. The applied force was minimized before each image acquisition.

The thickness of the PMMA layers obtained by spin-coating on silicon wafers was also evaluated using AFM. For this purpose, a 300 x 300 nm2 area was first imaged at high loading force; a larger image was then acquired, allowing to measure the depth of the previously damaged area.

The O/C and N/C molar concentration ratios determined by XPS are presented in Table 1.

The Ois and Cis spectra recorded on the three samples prepared without collagen were similar to those presented in the literature (Beamson and Briggs, 1992) and the O/C ratios were close to the value of 0.40 expected from the stoechiometry of PMMA. Adsorption of collagen led to N/C ratios of 0.12 and 0.08 at high and low drying rates, respectively, while the O/C ratios were not appreciably affected compared to the ONC ratios of samples conditionned with PBS. The Cis and Ois peaks were a combination of those of PMMA and collagen, the PMMA contribution being more important at low compared to high drying rate.

The surface composition of samples prepared by collagen adsorption, fast drying and subsequent spin-coating was dependent on the concentration of the spin-coated PMMA solutions: the N/C ratio was not significantly affected by spin-coating the solvent; it was approximately reduced by half at a PMMA concentration of 0.2 g/l, and it dropped by a factor of 10 at concentrations of 1.0 and 2.0 g/l. The Cis and Ois spectra changed progressively, with a decrease of the collagen contribution as the PMMA concentration increased. Spin- coating with PMMA solutions and even with the solvent alone on the samples slowly dried after collagen adsorption provoked the disappearance of the nitrogen signal. The Cis and Ois spectra were similar to those of pure PMMA, even when the pure solvent was spin-coated.

Table 1 : O/C and N/C molar concentration ratios determined by XPS on PMMA discs conditioned with collagen and subsequently submitted to PMMA spin-coating.

Collagen conditioning spin-coating molar ratio Concentration drying [PMMA] (g/l) O/C N/C (ug/mi) Oa FD none 0. 4t <0. 005 Oa FD 0.0 0.42 <0.005 Oa FD 2.0 0.41 <0.005 7 FD none 0.38° 0.123b 7 FD 0. 0 0.37b 0.113 b 7 FD 0.2 0.39 0.073b 7 FD 1.0 0.41 b 0.013b 7 FD 2.0 0.4Ob 0. 010b 7 SD none 0.38b 0.079b 7 SD 0. 0 0.45b <0.005 7 SD 0.2 0.39b <0.005 7 SD 0.5 0.37b <0.005 7 SD 1.0 0.40b <0.005 7 SD 2.0 0.39b <0.005 a Conditioning with pure PBS b Mean value of at least two independent measurements; the range of the data was not larger than 0.04 ° Mean value of three independent measurements; range of 0. 07 The thickness of the layer obtained by spin-coating with PMMA solutions of concentration of 2.0,1.0 and 0.5 g/I on silicon wafers was estimated, using AFM, to be respectively about 5,2 and 1 nm. At a concentration of 0.2 g/l, the layer was no longer continuous. The XPS data were modeled in order to confirm these results. The Si2p peak was decomposed into contributions of oxidized (Siox) and elemental (Siel) forms, and the thickness of the oxide layer present at the surface of silicon was estimated, using the Siox/Siel ratio, to be 2.0 nm

(parameters used: concentrations Cri. sien = 85.7 mmol/cm3, Csi. siox = 18.7 mmol/cm3 ; inelastic electron mean free paths Asi. sie) =si, siox= 2.9 nm (Mitchell et al, 1994)). The thickness of the PMMA layer was then evaluated using the ratio of concentrations of carbon involved in ester bonds and of elemental silicon, considering a continuous layer of PMMA over a 2.0 nm-thick silicon oxide layer, itself covering a silicon substrate (additional parameters used: concentration Co-s=o, pMMA = 11. 9 mmol/cm3 ; inelastic electron mean free paths AC, PMMA = 3.25 nm (Ashley, 1980) and AS§, PMMA = 2.9 nm). The PMMA thickness was found equal to 3.4,1.4, 0.8 and 0.5 nm at respective concentrations of 2.0,1.0,0.5 and 0.2 g/l, which was in the same range as the thicknesses measured using AFM.

AFM images obtained on the three samples prepared without collagen (Table 1) are presented in Figure 3. When no spin-coating was performed following conditioning with PBS, the PMMA surface was very smooth (r. m. s. roughness (Rrms) = 0.4 nm on 5 x 5 um2 areas) and defect-free. After spin-coating with the pure solvent or a 2.0 g/i PMMA solution, the surface was slightly rougher (Rrms =1. 1 nm on 5 x 5 um2 areas) and presented some holes or bumps.

Images acquired on samples fastly dried after collagen adsorption, before and after spin- coating with PMMA solutions of increasing concentrations, are shown in Figure 4, with cross- sections showing the characteristics of the relief obtained. The adsorbed collagen layer was homogeneous and smooth as shown by image a (Rrms = 0. 8 nm on 5 x 5 um2 areas). Spin- coating with pure chlorobenzene on top of this layer provoked the appearance of a rather regular array of holes as illustrated by image b. Quantification of the hole dimensions made on three independent samples, with three images each, gave a diameter of the order of 100 to 400 nm, a depth of the order of 10 to 50 nm, and a surface occupancy of about 23 %. After spin-coating with a 2.0 gui PMMA solution (image d), the surface relief was much more pronounced and had a different character. No clear baseline could be identified; the surface seemed to be covered by particles of a height of the order of 30 to 80 nm and a width of the order of 100 nm, which tended to aggregate and occupied about 63 % of the surface (dimensions obtained by analysis of three independent samples, with three images each).

The situations at 0.2 g/l, illustrated by image c, and at 0.5 and 1.0 g/i (images not shown), were intermediate between those at 0.0 and 2.0 9/l.

AFM images and cross-sections obtained on samples slowly dried after collagen adsorption, before and after spin-coating with PMMA solutions of increasing concentrations, are presented in Figure 5. The adsorbed collagen layer was discontinuous: it showed holes with a diameter of the order of 100-1000 nm and rims with a thickness of the order of 3 to 12 nm.

Spin-coating with PMMA solutions of concentrations from 0.0 to 2.0 g/l led to a surface architecture independent of the PMMA concentration; it showed cavities with a diameter of the order of 100 to 1500 nm and a depth of the order of 50 to 250 nm.

The topographies of the surfaces may be compared at different length scales, using 2D power spectral density analysis of the fast Fourier transform of the AFM images (Wieland et al, 2000). This is shown in Figure 6 (a) for samples fastly dried and in Figure 6 (b) for samples slowly dried after collagen adsorption. The Rrms of PMMA was low and increased only slightly and monotonously with the length scale. Adsorption of collagen followed by fast drying increased only slightly the Rrms compared to PMMA; Rrms did not vary appreciably with the length scale. After spin-coating with chlorobenzene, the Rrms was much higher and increased up to a length scale of about 300 nm, which corresponded to the size of the holes present at the sample surface. Spin-coating with a 2.0 g/i PMMA solution rendered the Rrms even higher at high length scale due to the observed aggregates. However, the variation of Rrms at length scales below 300 nm followed that of the samples prepared by spin-coating with pure chlorobenzene, showing that the lateral size of the surface features was in the same range.

The surface pattern obtained after collagen adsorption and slow drying (Figure 6a) led to a significantly higher Rrms at high length scale compared to the starting substratum; Rrms increased markedly up to a length scale of about 300 nm. Spin-coating still considerably increased the Rrms, which rose at length scales comprised between 0.1 to 1 um. The profile of Rrms as a function of the length scale was similar for spin-coating with chlorobenzene and with a 2.0 g/i PMMA solution, showing that the effect of the solvent overwhelmed the effect of the spin-coated polymer.

The surface organization obtained after adsorption of collagen, from a 7 ug/ml solution, on PMMA and either fast or slow drying has been investigated in a previous study (Dupont- Gillain et al, 1999). The N/C ratio obtained here after fast drying was almost identical to that found before. Modeling the adsorbed layer as a film of constant thickness (parameters used: concentrations CC, PMMA =59,5 mmol/cm3, Cc, collagen = 56, 9 mmoUcm3, CN, collagen = 19, 7

mmol/cm3 (calculated from the collagen sequence); electron mean free paths AC, PMMA = 3.25 nm (Ashley, 1980), Ac, protein= 3.5 nm, AN, protein= 3.2 nm (Dewez et al., 1997)) led to an apparent thickness of about 1 nm. The patterned surface produced at slow drying rate was shown to be a net, with holes of a diameter of about 100 nm: PMMA was demonstrated to be exposed at the outermost surface in the holes left by the collagen net. In the work presented here, the main trends are reproduced; however, the collagen layer obtained after slow drying resembles a layer with holes and thick rims, rather than a net. This is attributed to kinetics aspects of dewetting (Sharma and Reiter, 1996).

Here, different surface patterns have been obtained on a PMMA substrate, at the nanometer scale, by spin-coating with either pure chlorobenzene or PMMA solutions subsequently to collagen adsorption. The role of the adsorbed layer on the pattern formation is essential as no comparable topographical change were observed upon spin-coating on the PMMA substrate (Figure 3).

Spin-coating with pure chlorobenzene on the discontinuous collagen layer obtained by slow drying led to a surface topography laterally similar to that of the initial collagen layer, but with much higher and thicker walls. Collagen was no longer detected at the surface. This indicates that the PMMA substrate exposed in the holes was dissolved by the solvent and redeposited on top of the collagen areas, which served as a template. Spin-coating of PMMA solutions of increasing concentrations did not modify the surface relief significantly compared to that obtained with the pure solvent. The dissolution of the PMMA substrate thus overwhelmed the effect of PMMA deposition from the spin-coating solution, the amount of PMMA brought by the solution being negligible compared to that dissolved from the substrate. The solutions of 0.2 to 2.0 gui indeed allowed the deposition of 1 to 5 nm-thick PMMA layers on silicon, while the surface relief obtained here was 10 to 100 times higher. Spin-coating with a solvent of the substrate is sufficient to obtain chemically homogeneous PMMA surfaces presenting a surface roughness controlled by the organization of the previously adsorbed and slowly-dried collagen layer.

Spin-coating with pure chlorobenzene on the smooth collagen layer obtained by fast drying provoked the apparition of holes in the layer, but the N/C atomic concentration ratio remained unchanged. As compared with samples covered by a discontinuous collagen layer (slow drying), the thin but dense collagen layer obtained by fast drying limited PMMA dissolution.

Restructuration of the 1 nm-thick collagen layer alone may not account for the observed hole formation. Considering the surface occupancy by the holes (23 %), this would indeed have produced holes of a depth of 1.3 nm, while 8 to 40 times deeper holes were observed. The holes were thus formed by pit dissolution of the underlying PMMA. Redeposition of dissolved PMMA was not in the form of a homogeneous film ; if this was the case, the N/C ratio would have been reduced at least by a factor of 5.

Spin-coating with PMMA solutions of increasing concentrations on the smooth collagen layer progressively increased the surface relief compared to the one obtained with the pure solvent.

The surface pattern obtained at a PMMA concentration of 2.0 g/l may not be formed by PMMA particles (about 63 % of surface coverage) on a collagen layer. If this was the case, the N/C ratio would reach 0.05, while it was found equal to 0.01. Neither may the observed particles be made of collagen : the original collagen layer should indeed have a thickness of at least 20 nm to form such aggregates, and the resulting N/C ratio should be as high as 0.63 x 0.34 = 0.21 (0.34 is the N/C ratio expected for pure collagen ; the carbon concentration is similar in collagen and PMMA). Moreover, the deposited PMMA layer brought by spin- coating, which is equivalent to a thickness of about 5 nm as observed on silicon, cannot account for the formation of particles with a 30 to 80 nm size. The observed surface organization thus results from the combination of dissolution of PMMA from the substrate through the collagen layer, and deposition of PMMA by spin-coating; collagen is included in PMMA particles, which results in the weak, but not zero, N/C ratio.

While spin-coating is often used to planarize surfaces, it was shown here that it also allows producing nanometer-scale surface features. Planarization is favored by deposition of a thick polymer layer, using a solvent with low volatility and a low-molar-mass polymer (Bullwinkel et al, 1995; Gupta and Gupta, 1998). Apart from the solvent (chlorobenzene has a rather low volatility), the conditions chosen here were more favorable to structure formation: the PMMA had a very high molar mass; the concentration of the polymer solutions were very low (0.2 to 2.0 g/I) compared to those used for planarization (40 to 100 g/I), leading to deposition of a very thin layer. Moreover, dissolution of the substrate PMMA was either governing or contributing to the relief formation, which was not envisaged in other studies.

Different surface patterns were obtained, at the nanometer scale, by spin-coating chlorobenzene or PMMA solutions on collagen layers adsorbed on PMMA substrates.

Dissolution of PMMA from the substrate was shown to be a major factor in structure formation. However, the surface architecture (topography and chemical composition) depended on the collagen layer organization. In the case of the discontinuous collagen layer obtained by slow drying, redeposition of PMMA dissolved by chlorobenzene produced a chemically homogeneous surface with cavities in the range of 0.1 to 1 um diameter and 50 to 250 nm depth, which resulted from the organization of the initial collagen layer, acting as a template. The PMMA concentration of the spin-coating solution was not an important factor.

In the case of the smooth collagen layer obtained by fast drying, the surface organization resulted from a combination of dissolution of PMMA from the substrate and deposition of PMMA by spin-coating. Depending on the PMMA concentration of the spin-coating solution, different structures were obtained, from a pitted collagen layer to a PMMA surface covered by particles of PMMA incorporating collagen.

As the adsorption is a particular feature of the invention, said step will be disclosed in detail.

Collagen layers were prepared by adsorption on smooth poly (methyl methacrylate) (PMMA), rinsing, and drying in different conditions. The surface organization of these samples was observed with AFM, and their wetting properties were examined in dynamic conditions, using the Wilhelmy plate method. Depending on drying conditions, a great variety of structures were obtained, from a continuous layer to net-like structures with different mesh sizes in the submicrometer range. Comparison with results obtained on polystyrene and plasma-oxidized polystyrene allowed the role of different experimental factors (substrate, adsorbed amount, rate of drying) and mechanisms (substrate dewetting, collagen mobility, molecular interactions between substrate, collagen and water) to be clarified.

The purpose of the following is to investigate the possibility to create gradients in the organization of a collagen layer by taking advantage of the variation of thickness of the water film left at the sample surface before drying. This was produced by drying the samples in vertical position. The resulting organization of the collagen layer was observed using atomic force microscopy (AFM) and dynamic wetting measurements (Wilhelmy plate method).

Poly (methyl methacrylate) (PMMA; Aldrich, Bornem, Belgium; Mw = 996,000) substrata were obtained by spin-coating (time = 60 s, speed = 5,000 rpm, acceleration = 20,000 rpm. s~1) a 10 % (w/w) solution of PMMA in chlorobenzene (VEL, Leuven, Belgium) on PMMA plates obtained by compression moulding between two glass plates at 240 °C.

PMMA samples (size of about 20 mm x 15 mm) were immersed vertically (down to about 10 mm) in 6 mi of a 7 ug/ml solution of collagen in phosphate buffered saline (PBS), for 2 h at 37 °C. They were rinsed three times by removing the solution and adding water (produced by a MilliQ plus system from Millipore, Molsheim, France), which did not provoke dewetting. The samples were then dried under a nitrogen flow (fast drying, FD), or under 95% relative humidity (slow drying, SD). In the latter case, drying was performed with the samples in vertical position.

The samples were then cut in two pieces (20 mm x 7.5 mm; see Figure 7); one piece was characterized with AFM and the other was examined using the Wilhelmy plate method. For the sake of comparison, samples obtained by collagen adsorption followed by slow drying in horizontal position were also examined with AFM.

AFM images of the adsorbed collagen layers were obtained in air, in the contact mode, using a Nanoscope Ili microscope (Digital Instruments, Santa Barbara, CA, USA) equipped with Si3N4 triangular levers (Sharp microlevers from Park Scientific Instruments, Sunnyvale, CA, USA). The scan rate was 1 Hz. The applied force was minimized before each image acquisition. Imaging was performed at the bottom of the samples, as well as 4 and 8 mm higher (hereafter referred to as middle and top, respectively; see Figure 7).

Wetting with water (MilliQ), in dynamic conditions, was investigated using a DCA 322 equipment (Cahn Instruments, Cerritos, CA, USA). The measurements were performed at room temperature. The water container was closed with a lid, pierced with a small hole for the suspension wire; this ensured a relative humidity of 95 % 1 cm above the water surface.

Three cycles of immersion and emersion (advancing-receding) were performed at a speed of 50 um. s-1 and the contact angles ((3adv, irec) were recorded. Therefore, the effect of buoyancy was corrected, and the position scale X was referred to the distance between the bottom of the sample and the three-phases contact line, as described before (Tomasetti et al., 1998).

Figure 8 presents an AFM image of a collagen layer obtained after adsorption on PMMA in vertical position and fast drying, together with the variation of cos8 measured during repeated wetting cycles on the same sample. The AFM image shows that the collagen layer is smooth and continuous, as was observed for samples prepared by adsorption in horizontal position

and fast drying. Concerning the wetting behavior, Badv and 6rec of the first cycle were equal to 56 and 12°, respectively. The shape of the second and third hysteresis loops differed from the first one: E) ad, was initially close to (3rec, and then increased to reach a larger value (6adv = 66°) compared to the first cycle.

AFM images of collagen layers obtained after adsorption on PMMA and slow drying in horizontal or in vertical position are shown in Figure 9, together with the variation of cos6 measured during repeated wetting cycles on the sample prepared vertically. The structure obtained after adsorption and slow drying in horizontal position consisted in a collagen layer, about 3 nm thick, with holes of 50-150 nm diameter, surrounded by 5 nm-high rims. The structure obtained following adsorption and slow drying in vertical position depended on the position on the sample. A net-like structure, with a mesh diameter of 100-200 nm and a thickness of about 3 nm, was observed in the upper zone. In the middle, the mesh dimensions were larger (diameter: 300-500 nm; height: 5-7 nm). Finally, in the bottom zone, a structure close to that observed in the middle but with broken meshes was imaged. The wetting curves (Figure 9e) did not reveal variations along the height of the sample; Oadv and Oreo were equal to 72 and 12°, respectively. These values remained almost constant upon repeating cycles.

The structure produced by slow drying after collagen adsorption on PMMA was previously demonstrated to resemble a net, with PMMA being present at the outermost surface in the holes left by the collagen net. Figure 9 shows that the structure obtained on a sample dried vertically varies according to the height. This may not be attributed to accumulation of collagen in the bottom zone, as the samples were thoroughly rinsed and collagen adsorption is largely irreversible (Penners et al., 1981). It is attributed to the increase of the drying time from top to bottom, due to water drainage. Accordingly, the increase of the mesh size from the top zone (100-200 nm) to the middle zone (300-500 nm) is accompanied by increase of the net thickness (from about 3 nm to 5-7 nm). For these heterogeneous structures, the wetting behavior is expected to present a hysteresis, with advancing and receding contact angles governed by the low-and the high-energy phases of the surface, respectively (Andrade et al., 1985). eadv is similar to that of PMMA, while Orec is close to zero ((3adv and Orec of pure PMMA are respectively equal to 74 and 54°). Moreover, (3adv and srec are not affected by the size and the shape of the collagen domains, and the hysteresis loop shape is not

modified upon repeated immersion-emersion cycles. It appears thus that 6adv is determined by PMMA, while grec is determined by collagen.

Oadv recorded during the first wetting cycle on PMMA with a continuous collagen layer (Figure 8) was significantly lower compared to that measured with heterogeneous collagen layers.

This reflects a difference in the surface organization of collagen molecules in the dry state and indicates that the surface of the fast-dried sample is entirely covered by collagen. eadv of the subsequent cycles increased with the distance X of the three-phases contact line with respect to the bottom of the sample, i. e. with the time spent by the sample outside of water.

This variation of oadv may be due to retention of water by the hydrophilic collagen layer and its progressive evaporation upon emersion. At the end of the second and third immersions, 6adv reaches a value higher than that recorded for cycle 1, and similar to that of PMMA. This suggests that upon emersion, the collagen layer organization may changed from continuous to discontinuous. The emersed sample zones are indeed in the vicinity of the water surface, i. e. in a humid atmosphere, in conditions close to those used for the slow drying procedure which allowed producing net-like collagen layers.

These results show that the conditions of drying PMMA with an adsorbed collagen layer allow the structure of the collagen layer to vary over a large range: continuous layer (Figure 8), layer with holes (Figure 9a), net with different meshes (Figure 9b and c), net with broken meshes (Figure 9d).

The formation of collagen structures upon slow drying might be thought of by analogy with demixtion of two fluid phases consisting of nearly pure water and concentrated collagen solution (Billmeyer, 1971; Kim and Lloyd, 1992). However, the behavior of adsorbed collagen molecules is certainly different compared to that of collagen in the bulk of the solvent.

Collagen adsorption indeed relies mainly on the dehydration of the protein and the substrate surfaces, and it has an irreversible character. Moreover, the properties of the substratum influence greatly the structure obtained: net-like layers were found on PS, but not on PSox where a stronger collagen-substrate interaction was observed. On the other hand, collagen has a strong tendency to aggregate. At low pH, collagen is in the form of monomers in solution in a wide range of concentrations and temperatures. At pH near neutral, the formation of collagen aggregates is enhanced and increases with the temperature and the concentration (Piez, 1985; Murthy, 1984).

It is clear that the structure formation is related to the loss of water. A dewetting process typically comprises the following steps: (i) thinning of the liquid film by evaporation; (ii) pore formation due to ruptures in the thin film ; (iii) pore growth; (iv) coalescence of pores leading to a polygonal structure resembling a two-dimensional foam; (v) decay of the ribbons forming the polygonal structure, due to Rayleigh instabilities (Mertig et al., 1997; Sharma and Reiter, 1996). The possible relations between these mechanisms and the different structures observed for dried adsorbed collagen layers will be discussed now. On the sample slowly dried horizontally (Figure 9a), pore formation occurred and collagen was pushed ahead by the line between the wet and the dewetted zones, which led to the formation of rims around the pores. At the top of the samples slowly dried in vertical position (Figure 9b), the number and size of the dewetted and collagen-free zones was higher, but the structure was similar to that found on the sample dried horizontally. For the fastly dried sample (Figure 8), the rate of evaporation was so high that, either the density of dewetting nucleation was very high, or the collagen molecules had no time to rearrange and follow the progression of the line between the wet and dewetted zones.

The structure observed in the middle of the sample slowly dried vertically (Figure 9c) shows much larger meshes compared to the net-like structures already discussed. In this zone, the water evaporation is accompanied by a supply of water due to drainage. This might be responsible for a slower nucleation of dewetting leading to a lower density of dewetting pores and to larger holes in the collagen layer. An alternative explanation is that the amount of water present at the surface after pore formation is enough to allow collagen migration and, as a consequence, collagen further aggregation, thread rupture, and mesh coalescence. The last explanation is supported by the structure observed at the bottom of the sample (Figure 9d) which shows thread ruptures.

Knowing the exact nature of the mechanisms involved in the structuration of adsorbed phases is important to produce nanostructures with a well-defined designed. Key points are clearly the rate of drying, the adsorbate-substratum interactions, and the properties of adsorbate- adsorbate-solvent associations at the surface. Such mechanisms could be investigated further by extending the methodology used here, controlling the density of pore nucleation upon dewetting, the rate of progression of the lines between wetted and dewetted zones, and the amount of water remaining at the surface after pore formation. This could be realized by programming accurately the variation of humidity and temperature as a function of time.

Results obtained with PS and PSox indicate further that the substratum-adsorbate interactions and the adsorbed amount should be weak enough to leave sufficient mobility to the adsorbate.

Drying in different conditions of the collagen layers adsorbed on PMMA provided a great variety of structures, from a continuous layer, to net-like structures. The, gradients of collagen nanostructures obtained are of great interest in various fields, such as cell culture ; they indeed allow a direct comparison of properties as a function of the dimension of surface heterogeneities.

The organization of the collagen layer influenced strongly the macroscopic behavior upon wetting with water. All net-like structures gave an advancing contact angle determined by PMMA and a receding contact angle determined by collagen. The continuous collagen layers obtained by fast drying seemed to reorganize during the drying process occurring after an immersion-emersion cycle.

Comparison of the results obtained on PMMA with those obtained on PS and PSox allowed the influence of several factors affecting structure formation upon drying to be clarified. This structure is determined by a dewetting process, which is influenced by the nature of the substrate and the rate of drying, and by the migration of collagen at the surface. Collagen is pushed by the two-dimensional meniscus created between wet and dewetted zones.

However, its mobility is decreased if the interactions with the substrate are stronger, if the amount adsorbed is higher, and if the water content of the adsorbed collagen phase is lower.

According to these guidelines, nanostructures with defined and reproducible characteristics should be produced by (i) selecting the adequate substrate, in view of its wetting properties and interactions with collagen ; (ii) choosing the appropriate amount of adsorbed collagen ; (iii) finely tuning the drying conditions, to control the rate of dewetting and the variation of the amount and distribution of adsorbed water as a function of time.

Different surface architectures, in terms of topography and chemical composition, could be produced by changing the polymer substrate, playing with the adsorbed compound and its organization, choosing another solvent, modifying the spin-coating parameters,...

Several experiments were performed in which one or more parameters were changed. The results will be disclosed hereunder.

PMMA substrata were obtained as described above. Polystyrene (PS; obtained from BP, Zwijndrecht, Belgium ; reference PS05232) substrata were made using the same protocol, the spin-coating being performed from a 11.5 % (w/w) solution of PS in toluene (VEL, Belgium).

Collagen adsorption was performed following a procedure similar to that described above.

However, the rinsing step was slightly different: 2 ml of pure water were added to each well ; 3 ml of the liquid were removed; the sequence of adding 3 ml of pure water and removing 3 ml of liquid was then performed four times. A slow drying procedure was also adapted and performed as follows : after placing the wet samples in a vessel containing a saturated Na2CO3 solution, a drop of 200 pi of pure water was added on top of the sample, and broken by slight agitation in order to ensure a homogeneous coverage of the sample surface by a thin water film ; the vessel was then closed and the samples left for 2-3 days.

Spin-coating on the obtained patterned collagen layers was performed with different solvents.

When the substratum was PMMA, the solvents were chlorobenzene, dioxane and chloroform.

When the substratum was PS, the solvents were dioxane, carbon disulfide and toluene. Spin- coating was done using the following parameters: time = 60s, acceleration = 20,000 rpm, temperature = 20 °C. The spinning speed was varied (1000-3000-5000 rpm).

XPS spectra and AFM images were acquired as described above.

The O/C and N/C molar concentration ratios determined by XPS are presented in Table 2.

The spectra obtained on the untreated PMMA and PS substrata as well as on these substrata after spin-coating with solvents were similar to those presented in the literature for those polymers and the O/C values were close to the values of 0.40 and 0.00 expected from the stoechiometry of PMMA and PS, respectively. Adsorption of collagen led to the appearance of a nitrogen signal. Whatever the solvent and the speed used for spin-coating on the collagen template layer, the spectra were again similar to those of the pure polymers, the nitrogen signal having disappeared. This shows that part of the substrata has been dissolved by the solvents and deposited on the collagen pattern during the spin-coating process.

The morphology, observed with AFM, is shown in Figures 10 to 14 for different surfaces produced by (i) spin-coating untreated substrata with solvents ; (ii) slow drying of a collagen layer adsorbed on the substrata; (iii) spin-coating with a solvent on the collagen template layer. In all cases, no structure was observed after spin-coating the polymer without previous treatment with collagen.

Figure 10 shows the effect of spin-coating with chlorobenzene or dioxane on a PMMA substratum covered by a collagen template layer with large meshes (diameter up to 1 urn ; height: 9 nm). Spin-coating with chlorobenzene led to smoothening of the original structure, the height of the rims being reduced to 5 nm. The same trends were observed when spin- coating was performed with chloroform (images not shown). Spin-coating with dioxane led to the formation of PMMA walls of a height of about 100 nm, with a lateral distribution imposed by the collagen pattern.

Figure 11 shows the effect of spin-coating with dioxane on a PMMA substratum covered by a collagen template layer presenting smaller holes (diameter in the range 50-500 nm; height: 6 nm). The obtained surfaces presented rather deep wells (-60 nm) of a diameter similar to the wider holes of the collagen template layer ; the smaller holes of the collagen layer seemed to be filled by the polymer.

Figure 12 illustrates the use of different collagen patterns to obtain a variety of surface structures after spin-coating with dioxane. The collagen meshes were either large (same description as in Figure 10) or broken. This led to polymer walls (height of about 100nm), obtained after spin-coating, with a net-like pattern or a broken net pattern, as imposed by the collagen template layer.

Figure 13 presents structured polymer surfaces obtained on PS. The collagen layer showed holes of a diameter of 100-400 nm and a depth of 10 nm. After spin-coating with carbon disulfide, wells of a depth of 90-170 nm were observed. Spin-coating with dioxane led to smoothening of the original relief, with wells of a depth of about 2 nm.

Figure 14 presents the surfaces obtained after spin-coating with toluene on a PS substratum covered by a collagen net on PS. The obtained structures are walls of a height (3-5 nm) smaller than that found for the collagen layer.

The examples presented herein show that: (i) different solvents can be used to create a structuration by substratum dissolution and redeposition on a collagen template layer, (ii) a variety of structures can be obtained (walls or wells ; height or depth larger or smaller than the original collagen pattern) depending on the collagen template and on the selection of polymer and solvent.

Table 2: O/C and N/C molar concentration ratios obtained at different stages using XPS: untreated polymer substrata, substrata after spin-coating with solvents, substrata after adsorption of collagen and slow drying, substrata after treatment with collagen and subsequent spin-coating with a solvent. Substrata Collagen Spin-coating (solvent, speed) O/C molar ratio N/C molar ratio treatment PMMA NO---0. 44 0. 003 NO chlorobenzene, 5000 rpm0.41 <0.002 NO dioxane, 5000 rpm0.44 <0.002 YES 0. 29 0. 04 YES chlorobenzene, 1000 rpm 0.40 <0.002 YES dioxane, 1000 rpm 0.42 <0.002 YES dioxane, 3000 rpm 0.43 <0.002 YES dioxane, 5000 rpm 0. 40 0. 003 PS NO---0. 02 0. 003 NO dioxane, 3000 rpm 0.02 0.002 NO carbon disulfide, 3000 rpm <0.002 <0.002 NO toluene, 3000 rpm 0.01 <0.002 YES---0. 27 0. 11 YES dioxane, 1000 rpm0.10 <0.002 YES carbon disulfide, 1000 rpm 0.02 0.01 YES toluene, 3000 rpm 0. 01 <0. 002 The invention has an interest in several fields of application, in which a surface relief with controlled characteristics at the sub-micrometer scale may be an advantage.

(a) Tribology : modulating the rubbing properties of a substratum by controlling the surface nanorelief.

(b) Microfluidics : modulating the diffusion boundary layer or the shear rate near a substratum surfaces by controlling the surface nanorelief.

(c) Immobilisation of active components in a defined environment by creating open cavities with controlled characteristics at the surface of the substratum.

(d) Retention of active particles (capsules, vesicles, liposomes...) at the surface of a substratum: improving the retention by tailoring cavities which fit the immobilized particles.

(e) Control of the distribution of active components (enzymes, receptors, antibodies...) at the surface of a substratum.

(f) Triggering biosignals in cells by controlling the morphology of the cell-substratum interfaces.

(g) Making a coating around colloidal particles attached to a substratum.

(h) Improving the biocompatibility of polymer devices.

Several detailed possible uses of the invention are: Polymer materials : creation of a relief allowing specific rubbing or lubrication properties (cfr. a).

Organic coatings (paint...) : creation of a relief allowing better anchorage of the coating without modifying the optical properties of the substratum (colloidal shotting).

Nanoelectronics : -insulation of a network of nanoconductors made of carbon nanotubes (cfr. g), -modulating the surface relief of organic semiconductors.

Bioconversion and chemical processes: immobilization of a (bio) catalyst in pockets creating a well controlled environment (physicochemical, hydrodynamic) (cfr. b, c, d, e).

Biosensors: - immobilization of biocatalysts or receptors (cfr. b, c, d, e) - optimizing the interface between an optical electrochemical or piezoelectric transducer (substratum) and a signaling component (label compound, enzyme) (cfr. b, c, d, e).

Mammalian cell cultures (cell sorting, bioproduction...) : control of cell behavior (adhesion, cytoskeleton, gene expression) by a selective distribution of signaling molecules on the substratum, e. g. patch distribution of adhesion proteins and its optimization for focal contacts (cfr. b, c, d, e, f) - by the intrinsic effect of a relief at subcellular scale (cfr. f).

Biomedical devices: implants and extra-corporeal circulation devices with improved biocompatibility ; bags and catheters with improved hemocompatibility.

Although the description is directed to the use of PMMA and PS as a polymer and collagen as the template layer, several other possibilities can be chosen and these separate combinations may be found in the art. The specific choice of the materials to be surface modified, the template layer, the solvent and the specific method steps, i. e. the template layer deposition and the solvent application depend upon the required final surface structure.

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