PT105960A - PROCESS FOR DEPOSITION OF BIOMATERIALS IN WATER REPELLENT SUBSTRATES AND BIOMATERIAL RESULTS - Google Patents

Abstract

The present invention relates to a process for the deposition of biomaterials on surfaces involving the patterning of areas with different surface tensions at the surface of the substrates with high repellence to water, through a treatment that increases wettability, such as a treatment with ultraviolet radiation and ozone or plasma. Another aspect of the present invention relates to the substrates comprising areas with differences of surface tension, which present specific and controllable wettability properties, which are useful for the controlled deposition of solutions of biomaterials confined to the wettable regions, allowing for their individual manipulation and analysis. A third aspect of the present invention relates to the products or devices comprising biomaterials deposited on the substrates. The present process presents as an advantage the possibility of depositing independent combinations of biomaterials, such as proteins, genes, cells and culture media in independent regions of the same treated substrate, being consequently useful for the analysis of the performance of the biomaterials in various biological environments, and an added-value in the production of biomaterials in the fields of tissue engineering, regenerative medicine, cell biology, development of pharmaceutical drugs, and the monitoring of the administration of drugs and diagnosis.

Description

DESCRIPTION

PROCESS FOR DEPOSITION OF BIOMATERIALS IN WATER REPELLENT SUBSTRATES AND BIOMATERIAL RESULTS

TECHNICAL FIELD OF THE INVENTION The present invention relates to a process for the deposition of biomaterials on flat surfaces with high repellency to liquids, treated and designed substrates as well as products or articles comprising the biomaterials deposited on the treated surfaces.

BACKGROUND OF THE INVENTION The performance of materials for use in biomedical applications can be measured in the laboratory according to the cellular response in controlled environments. The development and improvement of expedited analysis techniques to track different combinations of materials, such as cells, bioactive molecules and culture media, is essential in a time-consuming and cost-related approach.

Rapid analysis technologies were initially developed and applied in the pharmaceutical industry (1). However, there are few expedient analysis systems available on the market to analyze biomaterials. As a pioneer in the creation of the first " library " of polymers, Joachim Kohn (2) points out.

Many of the platforms for expeditious analysis are now based on cell adhesion repellent surfaces where nano / micro-quantities of polymer can be deposited and are in contact with the same medium, after immersion of the entire platform in a culture medium (3). , 4). A disadvantage of this type of platform is that it is impossible to place different materials in contact with different cellular environments and culture media, limiting the study to the combinations of materials.

Another possibility to perform a combinatorial or multi-factorial analysis is to resort to the use of commercial well plates (5,6) which, although allowing the study of the different materials in different biological environments, use relatively large amounts of biomaterials, which are separated by rigid walls and high height.

In the techniques based on platforms where small materials are deposited, there are several methods to obtain them, which can be divided into direct and indirect techniques.

In direct printing methods, the use of a mask or a template is not necessary. These can involve contact, being divided into contact printing, contactless printing and bundle-based techniques (7,8). In contact prints, a tip (usually metallic) is used to apply a material or energy to a surface. Non-contact printing, also known as " inkjet " printing, is obtained by ejecting volumes in the order of the nanoliters of a microcapillary at specific positions on a surface. This technique, generally used to print ink on paper, was adapted for the expedited analysis of cell response to biomaterials using an agarose coated glass surface (9). Two types of collagen were used as " paint ", obtaining standards with 350ym in diameter. The use of gradients, that is, structures where a property is continuously varied throughout space, is another alternative for combinatorial studies of biomaterial-cell interactions. For example, the effect of the modulus of elasticity of three-dimensional hydrogels on osteogenic differentiation was studied using a continuous gradient of ultraviolet (UV) crosslinkable poly (ethylene glycol), where pre-osteoblastic murine cells were encapsulated (10). However, in such systems there is a risk of communication between cells which, in addition to being exposed to the same culture medium, may communicate with one another as there is no physical barrier separating them.

Considering patent documents in the field of biomaterial combinatorial studies, three distinct examples may be focused: (i) contact printing in which microhydrogels are deposited on a surface repellent to cell adhesion, (ii) the manufacture of micro-wells by lithography of flat materials " sobt lithography ", where biomaterials or cells can be deposited, and (iii) the two-dimensional study of analysis of the influence of topography on the cellular response.

Anderson created a method for the production of micro-matrices of polymeric biomaterials (11). The surfaces of the substrates have low affinity for cell adhesion, allowing to organize the polymeric biomaterials as elements of a micro-matrix on the surface of the substrate. In another approach of this method, a cellular encapsulation is made in the microarray of polymeric biomaterials wherein an appropriate dosage of cells is added to each microarray element. The substrate is immersed in a single culture medium. This prevents each of the biomaterials deposited in the microarray being completely independent of the rest of the microarray, so that all the samples have to be tested under the effect of the same cell culture medium. A further disadvantage of this method 3 relates to the volume of biomaterial liable to be deposited. Since there are no physical forces strong enough to prevent scattering of the biomaterials in the platform, the structural control of the biomaterials dependent on their height is restricted, for example making insertion of the porosity impossible.

Khademhosseini (12) has developed an expedited analysis device containing micro-array shaped micro-wells and a corresponding printed template for combinatorial analysis. The arrangement of the devices for depositing the biomaterials for rapid analyzes corresponds to the arrangement of the micro-wells, such that each reservoir addresses a single micro-well when the two micro-arrays come into contact. However, this is a processing that involves several steps, making it complex. In addition, as in commercial well plates, the materials are surrounded by walls and, because of their small size, it is also not possible to control the micro-environment in each region containing a biomaterial or a combination of biomaterials.

Another method (13) proposes a library of surfaces with distinct topographies generated through the use of mathematical algorithms. Surface generated libraries and topographies can be applied to understand the relationship between cell response and substrate relief in order to find surfaces with optimized or special performances. In this method the whole platform is immersed only in a type of culture medium, not having total independence between the regions with different topographies, which are only constructed in a two-dimensional logic. 4

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned problems, it is desirable to develop a method for depositing and combining different biological materials with different culture media and conditions, in a single substrate, according to claim 1.

In another aspect, the present invention aims to develop substrates useful for the application of said method, according to claim 4.

Another aspect of the present invention relates to products or articles comprising the substrates applied biomaterials according to claim 12.

Finally, in a further aspect, the present invention aims to develop the use of the above-mentioned articles in characterization analysis applications of the biological materials contained therein, as well as in tissue engineering applications, according to claims 13 and 14.

The present invention relates to a process for depositing biomaterials on substrates with high repellency to standardized liquids with wettable zones that allow the production of matrix chips of biological materials and media. Liquid volumes are confined in certain regions due to the strong contrasts of surface tension existing between the different regions defined on the substrate, thus allowing the independent, rigorous and controlled deposition of the desired biomaterials, biological materials, bioactive agents and culture media. 5 1. Substrates

In the context of the present invention, surfaces used as substrate for the deposition of the biomaterials should have water contact angles (WCA) greater than 110ø, more particularly 150ø. These contact angles allow the materials to be restricted to the wettable zone of the platform due to the contrast of surface tension with the water repellent zone which exerts a force contrary to the gravitational force which would allow the droplet to spread in the vicinity of the wettable zone.

Materials for the preparation of liquid repellent substrates may be based on natural or synthetic polymers, metals, inorganic materials or a combination thereof. After being processed, the substrates may undergo further treatments using chemical, physical, mechanical processes or processes involving the combination thereof.

Surfaces with contact angles above 150 ° are designated as superhydrophobic and must have topography showing roughness at the micro- and nano-scales. The liquid repellent substrate may have any geometry, including that of a regular and irregular, circular or ellipsoidal polygon, the most common being squares, circles or rectangles. The geometric variability allows manipulations in the substrate and deposited materials to be destroyed or modified, since they can thus be introduced in equipment with different geometries and dimensions.

Within the scope of the present invention, any liquid repellent surface may be used provided that it does not interfere with the chemical properties of the liquid formulation. Surfaces that are as inert as possible from the biological viewing point are preferable, so as to avoid interactions with the materials. This includes surfaces composed of polyolefins treated so as to have an increase of micro- and nano-roughness, as well as glass and ceramics treated with hydrophobic molecules or with high carbon coatings, such as carbon nanotubes. The preparation of highly repellent surfaces to liquids, namely superhydrophobic, is carried out by known techniques (14-15). In this sense, two approaches have been explored: (i) production of materials with surfaces with controlled texture structure and (ii) production of materials with surfaces of random structure.

In the former, the rough features are defined using techniques such as lithography or microfabrication. In the case of materials having a random surface structure, a wide variety of methodologies may be employed, including processes involving physical and chemical modifications of surfaces in order to obtain roughened surfaces. Examples of chemical and physical modifications to achieve roughness include coating of fluoropolymers layers, deposition of carbon nanotubes, phase separation, crystal growth, particle aggregation, vapor deposition, sublimation and " electrospinning ".

In particular, simple methodologies based on phase separation can be used to modify synthetic polymers on substrates with high liquid repellency. In a preferred embodiment of the present invention the treatment of surfaces is effected in order to increase its micro- and nano-roughness. This feature allows that, if desired, there is no subtancial induction of cytotoxicity of the surfaces. To increase their hydrophobicity, they can be immersed in 7 fluorinated solutions or with other molecules that induce greater hydrophobicity. 2. Substrate treatment and standardization

Within the scope of the present invention, the biological materials and / or combinations of biological materials and culture media are deposited in predetermined and treated regions of water repellent surfaces described above, taking advantage of the differences in surface energy existing between the regions of that surface.

In this sense, it is necessary to carry out the treatment of the surfaces in order to create said differences of surface tension ie it is necessary to standardize the substrate. In this way, the deposition of different combinations of liquid formulations, containing the biomaterials of interest, is carried out in the hydrophilic regions of the repellent substrate. The strong contrast between the surface tension of the wettable regions and the repellent regions of the platform prevents any leakage of the liquids out of the wettable regions.

For the modification of the superficial energy of surfaces there are techniques that allow the deposition of hydrophobic molecules, including the self-organization of monolayers of alkanotiools, organic silanes and fatty acids.

Substrates with high liquid or superhydrophobic repellency can be standardized using various surface treatment techniques, as previously described in other works (16-17). In this invention the wettable regions are preferably obtained using UVO or plasma radiation. Alternatively, still considering other surface modification techniques, methods such as Corona treatment or physical vapor deposition may be used. In this way, 8 regions are obtained through the placement of polymeric or metallic masks with geometrically empty / open regions. The mask only protects the regions of the selected repellent surface and thus the exposed regions of the open part of the mask are those that undergo surface treatment.

Alternatively, the obtaining of standard substrates within the scope of the invention is carried out by protecting regions of the substrate which are intended to be wettable with adhesive tape. Thereafter the surface treatment procedure is performed to increase the hydrophobicity, to transform the unprotected regions into a repellent surface.

The protected regions maintain the wettability of the untreated substrate. In case the surface to be treated is transparent the advantage of this methodology is to obtain transparent modified regions, which may be relevant in image analysis techniques.

The wettable regions can accommodate volumes of liquids due to the wettability contrast with the surrounding regions of the substrate. 3. Deposition of biomaterials

According to the process of the present invention, the biomaterials are in solution or suspended in a liquid medium which is deposited on the surface of the treated and standardized substrate.

Different combinations of liquid formulations may be deposited in different hydrophilic regions, defined by the treatment and standardization mentioned above, of the substrate. The strong contrast between the surface tension of the hydrophilic wettable regions and the hydrophobic repellent regions of the treated substrate prevents any leakage and mixing of the liquids containing solutions or suspensions of the biomaterials out of the wettable regions.

Droplets of liquid formulation are deposited onto the hydrophilic or superhydrophilic regions of the treated substrate.

Hydrophilic or superhydrophilic regions, also called wettable regions or regions, are surface regions having 0 to 80 ° water contact angles, preferably less than 50 °. Droplet deposition may be made using methods known in the art, including dispersion, atomization, electrostatic deposition, pipetting, jet-ink technology or other technologies which may generate liquid droplets.

In the context of the present invention, the biomaterials to be used must be dissolved or suspended in aqueous base solutions. comprise proteins, and synthetic polymers, as well as combinations of the

In this regard, the biomaterials polysaccharides, nucleic acids including polyesters or derivatives, referred to biomaterials.

Examples of polyesters are polycaprolactone.

Biomaterials are solubilized or dispersed in aqueous solutions, for example in the form of an emulsion. Other less conventional solvents, such as ionic liquids, may also be used, which enable the dissolution of substances such as cellulose, silk or chitin. As with the aqueous solutions, suspensions and saturated solutions may also be deposited therein. 10

Examples of ionic liquids are based on 1-alkylimidazole and 1-butyl-3-methylmidazole and 1-butyl-3-methylimidazolium.

Accordingly, solutions of any natural or synthetic polymer combined with low or high molecular weight molecules, such as genes, nucleic acids, peptides, drugs and growth factors, which are solubilized or suspended are encompassed within the scope of the present invention in aqueous solutions with pH ranges of 2 to 10 or in ionic liquids.

Examples of solutions are chitosan solutions in a concentration range of 0.5% to 4% (mass / volume) in acetic acid dissolved in water at a concentration of 1% (volume / volume) may be dispensed into the wettable zones of the platform by pipetting. Porous structures can then be obtained by lyophilization of the entire structure. The shape and extent of dispersion of the confined liquid in the hydrophilic / superhydrophilic regions depends on the volume of the deposited liquid and the wettability of the region. In this way, the present invention enables the deposition of volumes with a volume greater than the cube of the characteristic size of the region, due to the strong contrast of the surface tension of the treated substrate. The height control of the deposited biomaterials allows to work one more spatial variable in relation to the substrates of the prior art: the treated substrate according to the present invention makes it possible to obtain a structural control of the biomaterials.

Thus, it is possible, for example, to design porous structures that are compatible with cell migration and proliferation. In this case, each pore should be at least 100 Âμm in diameter, with a number of pores significant in relation to the volume of the structure itself.

In addition, the possibility of height control of the structure allows the study of interfaces between biomaterials arranged in layers, of which height and composition may vary. It should also be noted that cells in most human tissues are in three-dimensional environments (extra-cellular matrix) where the ratio of width, length and height in general is similar. This is not the case in the substrates of the prior art, since even when the deposited materials are considered three-dimensional, their height is generally much lower relative to the dimensions of the material in direct contact with the substrate (width and length). The volume of the droplets may vary between 0.5 pL and 15 pL, depending on the size of the wettable regions. It has now been found that these values correspond to the minimum and maximum for the dimensions of the wettable regions considered for the present invention (200 pm-3 mm).

These values are reduced enough that the application may have advantages in terms of space and quantity of material used over conventional methods, such as processing of materials in commercial well plates. However, they are volumes high enough so that the samples can be structurally controlled in terms of porosity or representative height relative to the wettable regions. The size of the wettable regions may be dimensioned intermediate between the dimensions of the materials obtained by the techniques described in the art relating to the manufacture of microarrays and the size of the wells of the commercial cell culture plates generally used for individual analysis of biomaterials.

In this way, the substrates of the present invention may be used in broader applications than the existing modified substrates in the art or that commercially available culture plates.

The dimensions of the wettable regions between 200 and 3 mm, and preferably between 400 and 1.5 mm, may simultaneously allow substrates with a high number of deposited biomaterials to be obtained and the individual regions individually manipulated, analyzed and characterized. The space margin between each wettable region should be greater than 500ym and less than 3mm, as it allows an efficient separation of the materials, avoiding their junction due to electrostatic affinity or some inclination resulting from the handling of the substrate. The contrast of wettability between the wettable regions and the repellent zones enables the treated surfaces or substrates to be handled, including their inclination, without mixing the deposited elements in the wettable regions.

The biomaterials in the liquid portions adhere to the surface in the wettable regions by adsorption, evaporation / separation of the liquid from the solvent, cross-linking or by another insolubility inducing method. 4. Products or articles obtained

The products or articles of the present invention result from the deposition of the biomaterials in the wettable zones of the subtrees treated in accordance with the present invention. 4.1 Two-dimensional materials

From a two-dimensional analysis perspective, biomaterial films can be obtained by the successive deposition of polyelectolites, by the technique known as " layer-by-layer " (LbL) or layer on layer, to the surface of the substrates treated in accordance with the present invention. LbL technology allows the production of nano-structured coatings using poly-electrolytes with opposing charges (negative and positive). For this purpose a sequential deposition of poly-electrolyte solutions with opposite charges is intercalated by washing with an aqueous base solution, for example a solution of 0.1 M NaCl in distilled water. At the end, films of water-insoluble biomaterials, bound merely by electrostatic interactions, without the use of other compounds are obtained.

The present invention provides for the deposition of cell suspensions in films and coatings produced by different combinations of polymers, especially polysaccharides (including chitosan, hyaluronic acid, heparin, alginate or chondroitin sulfate).

In this way and in accordance with the present invention, it is possible to carry out the deposition of different polyether polyols in multilayers of different thicknesses, it being possible to carry out numerous possibilities of combinations of materials, number of multilayers and processing variables (eg ionic strength, pH, polyelectrolyte concentration, subsequent cross-linking of the obtained film).

With respect to materials deposited two-dimensionally on treated substrates, the present invention provides for the deposition of biomaterials of different origins, such as, for example, composite biomaterials resulting from combinations of polymers with inorganic or metallic phases (such as nanoparticles).

Thus, it is possible to effect the deposition of liquids containing the two elements in the wettable regions of the platform and proceeding to the solidification of the nanocomposite (for example, by evaporation or sublimation of the solvent) and, if necessary, further treatment (e.g. neutralization).

In addition to macromolecular systems, the deposition of other materials is provided within the scope of the present invention, for example materials of interest in the study of the cellular response. For example, peptides, enzymes, self-organizing low molecular weight polymer molecules (resulting in low molecular weight hydrogels), and combinations of low molecular weight structures may be deposited as a solution / precipitate in the hydrophilic regions, followed by deposition of cell suspensions. Deposition of antibodies in the wettable regions (for example, by covalent attachment to the platform) to investigate adhesion of different cell types may also be performed using different combinations of antibodies and cell types. 4.2 Three-dimensional materials

In addition to interactions in a two-dimensional environment such as those described above between cells and preadsorbed proteins or two-dimensional films, in the scope of biomaterial development it may also be desirable to study materials in three-dimensional form, since they mimic more rigorously the environment in which the cells are under physiological conditions: the extra-cellular matrix. The deposition of biomaterials in the wettable regions of the substrate according to the present invention can be done so that after processing the final biomaterials have acquired a three-dimensional configuration including porous structures or hydrogels suitable for tissue engineering and cell culture , by techniques such as, for example, lyophilization, particle leaching or cross-linking.

The three-dimensional constructions are mechanically restricted in the hydrophilic / superhydrophilic region of the substrate because of the high liquid repellency of the surrounding regions. It is intended that after this processing the biomaterials be tested for tissue engineering applications, development of biodegradable and non-biodegradable implants, release of bioactive agents and coatings of implant materials. Application of the treated surfaces according to the present invention, in the study of protein adhesion and corresponding cellular response, the application relates to surface modification of biomaterials. When a material is implanted, the first occurring reaction is the adsorption of proteins to the surface of the material, to which the cells will later bind through the integrins present in their cell membrane, which may recognize peptide domains of the adsorbed proteins. Thus, pretreatment of two-dimensional or three-dimensional biomaterials with proteins with cell adhesion domains (such as human fibronectin (HFN)) is a means of increasing the affinity of a biomaterial with the surrounding cells, enhancing an easier and more efficient formation of fabric. On the contrary, the low adhesion of cells related to blood coagulation (such as platelets) may be desired in some types of biomaterials, such as vascular grafts. In this case, adsorption studies of proteins or other molecules that potentiate the low adhesion of these cell types may be carried out. 4.2.1 Porous structures

Porous structures for tissue engineering or cell culture are biomaterials where porosity is induced. The pores must be interconnected and have sufficient size (diameter greater than 100 μπι), in order to allow cell migration and proliferation within the structure.

In the case of porous structures, aqueous base solutions with monomeric or polymeric precursors may be deposited in hydrophilic / superhydrophilic regions in volumes ranging from 0.5 pL to 15 pL. These structures can be obtained by a variety of methods, including lyophilization and cross-linking, using combinations of natural or synthetic polymers in solution, including polysaccharides, polyesters and proteins. The first contact of the cells in a physiological environment with a biomaterial is with its surface. However, there is not always a balance between the structural properties of the implantable biomaterial and its surface properties. Thus, in order to achieve the desired effect on cell behavior, surface treatments are optionally carried out by protein adsorption, chemical bonding of favorable chemical groups or inhibitors of cell adhesion, physical treatments for induction of desired topography, among others.

Subsequent treatments to the porous polymeric structures can be achieved by introducing other liquid volumes into each of the structures, such as by adsorption of proteins in solution. In such treatments the insertion of peptide domains of cellular adhesion or the chemical or topographical modification for the increase of the adhesion, proliferation or some specific type of cellular response is usually sought.

Other methods for obtaining porosity in the polymeric structures, in addition to lyophilization, may be the introduction of leachable particles into the initial formulation. Inorganic (e.g., sodium chloride or sugar) or polymeric (e.g., poly (caprolactone)) particles with pore size ranges may be introduced into polymeric formulations. During immersion of the substrate in specific solvents for the particles (eg water in the case of sodium chloride or sugar particles) these are removed leaving only the insoluble polymer fraction, which will ultimately have a porous three-dimensional structure. The size and amount of pores are controlled by the size and amount of particles to be leached.

In another aspect of the present invention, solutions of polysaccharides or other methacrylated polymers (e.g., chitosan or dextran) may also be deposited in the hydrophilic / superhydrophilic patterns, which are then cross-linked and fixed by ultraviolet radiation. This biomaterial processing method allows for greater compatibility with cellular encapsulation, absence of toxic products presented, for example, by most chemical crosslinkers, and even greater reproducibility and speed to obtain the biomaterials in their final form. 4.2.2 Hydrogels

Hydrogels are materials composed of hydrophilic polymer chains, which when crosslinked form a network with high water absorption capacity. Biomaterials in hydrogel form can be prepared using chemical and physical crosslinking methods. In this invention, the preparation of the hydrogels 18 is preferably carried out after deposition of the monomer / polymer precursor in the hydrophilic / superhydrophilic pattern of the substrate, as described previously for the porous structures. The deposition is followed by cross-linking of the monomer / polymer solution. These structures are useful in the development of implantable biomaterials and release of active agents. Various methods are known for obtaining hydrogels, among which are crosslinking obtained by chemical agents which lead to the formation of covalently linked groups, exposure to ultraviolet radiation of polymers modified with methacrylate groups, ionic gelling or cross-linking by temperature variation.

As an example of hydrogels are alginate hydrogels using solutions of alginic acid in water in ranges of 1% to 3% concentration, ionically crosslinked with various concentrations of calcium chloride in concentrations of 1 M to 10 M.

The deposited hydrogels may also contain encapsulated cells, i.e., cells may be contained in the polymeric or monomeric precursor of the hydrogels prior to their processing. In this case it is necessary to use crosslinking forms which maintain cell viability, as in the case of crosslinking of methacrylate polymers, for example in the case of polysaccharides such as chitosan, alginate, hyaluronic acid or dextran, by exposure to ultraviolet or visible radiation. Another possibility compatible with cellular encapsulation is ionic gelation, which can be obtained, for example, by crosslinking alginate with calcium ions. Also materials which gel upon temperature variations, for example gelatin-based, can be used in applications involving cellular encapsulation. 19

The mechanical properties of the hydrogels and their water adsorption capacity can be controlled by varying the exposure time to the crosslinker or the amount / intensity thereof in each of the hydrophilic / superhydrophilic regions. Different polymers may be blended in the same hydrophilic / superhydrophilic region, including naturally occurring polymers and synthetic polymers.

In each wettable region more than one polymer may also be deposited sequentially so that there is no mixing between polymers resulting in layers of biomaterials in the same wettable region.

In the case of hydrogels, structures with more than one polymer may be obtained, for example, in the form of semi-interpenetrated networks or interpenetrated networks. The process of the present invention has application in the analysis of the deposited biomaterials in the treated and standardized substrates and in general terms this analysis can be classified into two types: physical-chemical analysis and analysis of the biological response. In both cases, modifications or adaptations to equipment originally prepared for analysis of individual samples may be required. Preferably, the samples should be analyzed on the platform, without their removal and destruction being necessary. 4.3. Biomaterials analysis

As for the physical-chemical analysis of biomaterials, in the case of three-dimensional porous structures, morphological analysis and porosity quantification can be achieved using different methods of structural analysis, including scanning electron microscopy and computerized microtomography. 20

For a mechanical analysis of the elements deposited on the platform, methods such as static compression tests, nanoindentation and microindentation can be used. The viscoelastic behavior of the samples can be analyzed by dynamic mechanical analysis, either under dry conditions, or in situations where the sample is immersed in a suitable liquid medium.

Also for porous structures and hydrogels, the chemical composition and distribution of the materials in the structure are determining factors for the cellular behavior. For an analysis of the atomic composition of the structures, the dispersion of X-ray energy can be used, for example.

For a mapping and chemical characterization of the structures without recourse to the destruction of the platform, one possibility is the use of infrared Fourier transform spectroscopy in microscopic mode. In this equipment, the platform is placed on a table with possible movements in the xyz axes, and an infrared beam is attached to the sample. The transmitted beam is then analyzed and the type of chemical bonds in the analyzed portion of the structure, represented by the intensity of infrared transmission peaks, can be accessed.

For the analysis of the biological performance of biomaterials, two possibilities can be considered. In the case of porous structures, the cells are deposited on top of the processed biomaterial in its final stage on the platform in each of the hydrophilic / superhydrophilic patterns. In the case of hydrogels, the same procedure can be performed, or the cells can be directly encapsulated in the hydrogel during their processing on the platform.

To ascertain the cytocompatibility of the materials, such as their ability to withstand cell proliferation and migration, cell lines can be used, for example 21 SaOs-2, fibroblast L929 or chondral MTDC5 pre osteoblastic lines.

For an approximation to the actual behavior of the biomaterials in contact with cells and tissues, primary or stem lines may be used, as well as co-cultures. The process of the present invention also enables the use of more than one type of cells in contact with each of the materials deposited on the platform if the biomaterials are composed of layers of different materials.

In the context of this invention, the analysis of the cellular behavior will preferably be done by techniques based on image processing, in order to avoid the destruction of the platform and so that the automation of the image acquisition allows a rapid analysis of the results.

For a preliminary analysis of viability, metabolic activity and cell proliferation, techniques such as labeling with Calcein AM and " 4 ', 6-diamidino-2-phenylindole " (DAPI) can be used in two-dimensional and three-dimensional samples. As controls, quantification of " 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) " (MTT) or " 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium " (MTS) / Alamar Blue and dsDNA, respectively, can be performed. While in the viability / metabolic activity study the cells must be analyzed live, in the proliferation study, these can be analyzed live or fixed. Cell morphology can be studied by scanning electron microscopy or confocal microscopy.

In a differentiation analysis, taking as an example osteogenic differentiation (although the invention is not restricted to this type of differentiation), after 21 days or more of cell culture, analysis of osteogenic markers may be performed by immunofluorescence . Markers such as collagen type I, osteocalcin and alkaline phosphatase can be studied.

Coloring studies for the search for phenotypic differentiation markers, such as calcium (using Alizarin red) and phosphates (Van Kossa stain) may also be used and the results observed by optical microscopy, without the need to remove structures from biomaterials of the substrate of the invention.

By avoiding immersion of the entire substrate in a well with culture medium, it may also be possible to study different culture media, namely media with different additives such as drugs, toxic molecules or proteins, in each platform independent structure. It is also possible to vary the biological environment in contact with biomaterials over time by aspirating / replacing media from distinct cultures in two-dimensional or three-dimensional structures. Further studies will be the same as those listed above.

Brief description of the figures

Figure 1. Representative image of SEM for smooth polystyrene surfaces (A) and rough with extreme liquid repellency (B). The micro- and nano-structure of the superhydrophobic surfaces was obtained by the phase separation methodology consisting of dissolving the polymer with the tetrahydrofuran solvent, subsequently mixing the solution obtained with a non-solvent (ethanol) to force the precipitation of the polymer. The precipitation of the polystyrene leads to the formation of the rough surface through the formation of nuclei that causes the decrease of the surface tension.

Figure 2. (A) Schematic sequential representation used to produce substrates with extreme liquid repellency, standardized with wettable regions to be applied in tests of interaction between cells and proteins. (B) Water droplets with different volumes from 2 pL to 8 pL confined in the wettable regions produced by different UV / Ozone (UVO) times from 1 to 12 minutes.

Standard liquid repellent surfaces with wettable regions were produced using 1x1 mm2 open-space masks (Figure 2A). The shape and dispersion of the confined liquids in the wettable regions depends on the volume of the liquid deposited and the wettability. Figure 2B shows that for regions with the same wettability using volumes greater than 6 pL, the liquids remain confined in the wettable zones because of the strong difference in surface tension with the surrounding repellent zones.

Figure 3. (A) Measurement of the contact angles of the evolution of wettability as a function of the UVO irradiation time for smooth and rough polystyrene surfaces. The images show water droplets on top of the rough surface (left) and smooth (right) on three different levels of exposure to UVO radiation. (B) Spectra XPS Cls of the superhydrophobic surface (spectrum above) without exposure to UVO radiation and of the same surface after 12 minutes of exposure to UVO radiation (spectrum below). Modification of surface wettability was achieved by surface exposure to UV / Ozone (UVO) radiation. The rough surface spectrum is divided into two peaks corresponding to C-H and C-O groups which are centered at 285.01 eV and 286.30 eV. After 12 minutes of UVO radiation, two more hydrophilic groups C = 0 and 0-C = 0 corresponding to peaks 287.90 eV and 289.26 eV respectively are detected. The results are consistent with the photochemical modifications of the -CH 3 groups of the roughened surfaces into CHO, COOH and OH groups. The topography of rough surfaces observed by SEM (Figure 1B) shows no changes with UVO irradiation. However, the observed changes in contact angles (Figure 3A) are the result of increased hydrophilicity of the material during UVO exposure, due to the introduction of the oxygen-containing groups on the surface.

Figure 4. Adsorption of human serum albumin, HSA (A) and human plasma fibronectin, HFN (B) after 2 hours of incubation in solutions with different concentrations of protein. The different symbols are defined as a function of the UVO irradiation time. The open symbols correspond to the roughened surfaces, and the filled symbols correspond to the conventional polymer surface which serves as the control (untreated surface). The 31 ° kinetic adsorption of HSA (C) and HFN (D) on rough surfaces after 6 minutes of UVO radiation using solutions with different concentrations of proteins.

The results indicate that there is a large amount of protein adsorbed in the first 30 minutes, following a very stable adsorption up to two hours of contact. The adsorption of proteins in the conventional polystyrene shows quite similarities with the rough polystyrene at the same contact angle (after 3 minutes), showing that the topography of the surfaces with the same wettability does not have a significant influence on the protein adsorption. For surfaces with extreme wettability, the amount of adsorbed protein is higher for surfaces that exhibit greater liquid repellency, showing that on protein-free (superhydrophilic) surfaces protein adsorption is scarce.

Figure 5. (A) Image obtained by fluorescence microscopy of the surface with extreme liquid repellency standardized with wettable regions, where 4and L of solutions of (Ai) HSA and (Aii) of HFN with different concentrations (vertical axis) were deposited during different adsorption times (horizontal axis 25). (Aiii) Different combinations of HSA and HFN were deposited in the wettable regions of the liquid repellent surfaces where different relative amounts (vertical axis) and different concentrations of protein (horizontal axis) were used. (Bi) Image obtained by the confocal microscope of SaOs-2 cells cultured for 4 hours in the wettable regions of the platform with different amounts of preadsorbed proteins (equivalent to the micro-matrix Aiii). (Bii) Map of intensity map for the number of cells in each wettable region that corresponds to the same matrix tested with different concentrations of protein. Scale: 500 ym.

Different solutions were deposited with different relative amounts and concentrations of HSA / HFN. The sample on the treated substrate containing pre-adsorbed protein deposition was washed with buffer solution at physiological pH and observed under a fluorescence microscope. The control column was analyzed without pre-adsorbed protein where different numbers of cells were deposited. The number of cells fixed at each point is higher for larger amounts of pre-adsorbed HFN, or on surfaces where the relative amount of HFN in the HFN ratio / HSA is higher.

Different polymeric biomaterials were also tested using different volumes of solutions in the wettable regions of the repellent surface (Figure 2B). This platform can be integrated in tests that involve a great amount of substances to be tested under different biological conditions.

DETAILED DESCRIPTION OF THE INVENTION 1. Treatment and standardization of water repellent surfaces 26

Within the scope of the present invention superhydrophobic surfaces are treated and standardized in order to obtain regions having different surface tensions on the same surface. The standardization of surfaces with high water repellency with wettable regions is accomplished by the configuration of a mask on the superhydrophobic substrate with open zones for exposure to the treatment of the surface of the selected substrate and definition of zones with different surface tensions through UVO irradiation or plasmas. The UV radiation exposure time varies normally between 0 and 14 minutes. The surface modification can also be performed with Argon plasma at a power of 30 W and a pressure of 0.2 mbar, varying the exposure time of each section of the surface between 0 and 150 seconds

The applied masks define different dimensions and geometric shapes, said dimensions and shapes being drawn by placing adhesive tape or other treatment resistant film to increase the hydrophobicity in the areas to be protected, i.e. in the untreated zones.

The regions defined by the mask preferably have a dimension between 200 μm-3 mm, more preferably between 400 μm and 1.5 mm or even more preferably between 0.5-1 mm. 2. Preparation of Biomaterials Solutions or suspensions are preferably prepared with an aqueous basis of biomaterials which may include proteins, polysaccharides and polymers and / or combinations thereof. 27 3. Deposition of biomaterials

Droplets of solution or suspension of biomaterials having dimensions between 5 and 15 and 15 and L are deposited in the wettable regions, for example by micropipetting or other known technique to generate and deposit liquid droplets on a surface.

To obtain two-dimensional films, deposition of the biomaterials can be done by LbL technique.

In another preferred embodiment of the invention, the biomaterials deposited on the substrate are treated by lyophilization, leaching or cross-linking techniques.

After treatment to obtain its final form, the material deposited on the treated substrate is then preferably immersed in cell culture medium in a low CO2 atmosphere at temperatures between 30 and 40øC, more preferably between 35 and 38 And even more preferably at +/- 37 ° C, in an atmosphere containing less than 15% CO 2, preferably less than 10% CO 2 and still more preferably less than 5% CO 2.

Optionally, the biological material is fixed by addition / exposure to an organic solvent (such as formaldehyde or glutaraldehyde) and / or stained with material suitable for the biological product in question, for tracking and identification thereof. 4. Characterization of products or articles obtained

According to the process of the present invention two-dimensional structure products are obtained, resulting from the deposition of biomaterials on substrates treated in accordance with the present invention, using, for example, a micropipette.

In a preferred embodiment of the invention products having a bi-dimensional structure include films obtained by solvent evaporation or by LbL.

In another aspect of the present invention, three-dimensional porous or hydrogel structures are obtained by lyophilization, leaching or cross-linking techniques.

In a preferred embodiment of the invention, the hydrogels comprise the encapsulated biomaterial, such as encapsulated cells.

In another preferred embodiment of the invention, the hydrogels comprise encapsulated bioactive agents, such as, for example, drugs, growth factors, nucleic acids, peptides or genes.

In another preferred embodiment of the invention, the porous products include bioactive agents mixed with the precursor of the biomaterial prior to its processing which confers the porosity. The bioactive agents may also be immobilized or adsorbed after processing the biomaterial as a porous structure. Included are bioactive agents such as growth factors, drugs, peptides or proteins. Preferably, suspensions with cells should be deposited on the porous products after their final processing. 5. Application of products or articles

Substrates treated according to the process of the present invention have applications in areas of development of biomaterials since they enable the deposition of different combinations of biomaterials and liquid media.

In particular, the treated substrates of the present invention are especially useful in applications requiring combinations of biomaterials and culture media.

In this way, and due to the different combinations between biomaterials and different media in which they are in solution or suspension, when deposited on the substrates treated in accordance with the present invention, they have characteristics that differentiate them from the products or articles included in the state of the art, which only include individual combinations of a biomaterial with a culture medium applied to the surface of the substrate.

In a preferred application of the products of the present invention, use is included for rapid and expeditious analysis of the chemical, physical or biological properties of the materials.

In another preferred application of the present invention, application in bioengineering is included. In yet still more preferred application of the present invention, the development of implantable biomaterials or active agent releasing materials is included.

Examples

Example 1 - Obtaining superhydrophobic surfaces

Within the scope of the present invention surfaces of different polymers have been treated, obtaining at the end of the treatment a contact angle greater than 110Â °, preferably greater than 150Â °. Two polymers were used: polystyrene and lactic polyacid. 30 1.1 Polystyrene surfaces

The high water repellency surfaces of polystyrene were obtained using polystyrene plates cut from commercial Petri dishes, with average original contact angle of 98.9 °. These were treated with a solution composed of polystyrene dissolved in tetrahydrofuran (polystyrene solvent) at a concentration of 70 mg / mL, to which ethanol (non-solvent of polystyrene) was then mixed in a phase separation reaction.

After spreading the solution on the initial polystyrene plate, the solution is withdrawn and the entire plate is immersed in pure ethanol. The treated plate is then dried under a stream of nitrogen. The surfaces obtained had contact angles of more than 150 °, in particular with an average value of 156.2 °. The analysis of the surfaces treated by scanning electron microscopy showed the formation of hierarchical structures at the micro- and nanoscales: beads with diameters between 50 nm and 200 nm agglomerated in larger structures could be observed. 1.2 Surfaces of poly lactic acid

High water repellency surfaces were also obtained by reversing phase reactions using a biodegradable polymer, such as poly (L-lactic acid) (PLLA). Lactic polyacid grains were heated above their melting temperature (173-178 ° C) while being compressed in order to obtain homogeneous polymer films.

To increase its water repellency, mixtures of PLLA with 10% (mass / volume) dioxane were spread on PLLA plain films and after a few seconds the samples were immersed in pure ethanol so as to cause a phase separation. After total drying of the beads, the film coating the surface was removed and a structure with micro- and nano-roughness was obtained, bringing the surfaces to be presented with a mean contact angle of 153.6 °.

Example 2 - Treatment and standardization of super-

2.1 By irradiation with UVO

For the standardization of surfaces with high water repellency with wettable regions, a plastic photo-mask with open squares 1x1 mm2, 1 mm apart, was used. The UVO radiation exposure time was varied from 0 to 14 minutes, with contact angle values being recorded in all the minutes, allowing a variation of the surface contact angle of approximately 156 ° (corresponding to 0 minutes exposure to UVO radiation) at approximately 0 ° (corresponding to the exposure of 14 minutes to UVO radiation).

After treatment of the surfaces with different exposure times to UVO radiation, the Sessile drop method was used again to verify and quantify the decrease in the contact angle of the surfaces. In addition, X-ray photoelectron spectroscopy (XPS) was also performed at a 90 ° angle to the sample, with a constant energy of 100 eV for general analysis and 20 eV for high resolution analysis. This analysis allowed to observe the reduction or increase of functional groups in the polystyrene sample that are formed with the superficial treatment with UVO radiation. 2.2. By application of argon plasma The surface tension gradient of the selected substrate was obtained by treatment with Argon plasma at a power of 30 W and a pressure of 0.2 mbar.

In order to obtain the wettability gradient, a mask was slid across the length of the PLLA sample with high water repellency, varying the exposure time of each section of the surface to the surface treatment. Thus, the exposure time to plasma was varied from 0 to 150 seconds, and within that range of exposure times a contact angle variation of 153.6 ° to 0 ° was observed.

Example 3 - Preparation of solutions or suspensions containing biomaterials

Within the scope of the present invention, the adsorption of proteins present in human plasma was tested on the wettable regions of polystyrene substrates treated with UVO radiation through masks, in particular plastic films, with open regions of 1 mm2 for 6 minutes.

Human serum albumin (HSA) and human plasma fibronectin (HFN) solutions were prepared in buffer at physiological pH at concentrations of 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL and 50 mg / ml. HSA was suitably fluorescent green fluorescently labeled with isothiocyanate. HFN was labeled in the laboratory with " kit " protein markers Alexa Fluor 555.

Example 4 - Deposition of biomaterials on treated substrates

Different combinations of biomaterials and culture liquids were individually deposited and limited in the wettable (hydrophilic) regions, as a result of the strong wettability contrast between the initial sample and the modified regions, as seen in Figure 2B.

Repellent surfaces were standardized using UV / Ozone (UVO) (Figure 3) in order to modify surface wettability. The amount of protein adsorbed on surfaces with different wettability (Figure 4A-B) was evaluated to rapidly analyze the effect of protein concentration and adsorption time (Figure 4C-D). Other parallel adsorption studies of HSA and HFN were also carried out on substrates with different wettability among the superhydrophobic-superhydrophilic range.

Different combinations of HSA and HFN were adsorbed on the wettable regions of the platform, as seen in Figure 5A i-ii. Quantification of protein adsorption on control surfaces (diameter 13 mm) alone was performed by the Bradford method. In this case, only isolated adsorption studies were performed, ie on the same surface only HSA or HFN existed, and never both simultaneously.

Assays performed on the control surfaces with 6 minutes exposure to UVO radiation were reproduced in the wettable regions of the platform for expedited analysis within the scope of this invention. In this case, competitive studies of protein adsorption, in which HSA and HFN were placed in different ratios in the wettable regions, were also performed. Observation of stained proteins with fluorescent molecules reflected at different wavelengths (corresponding to green color and red color, respectively) was performed using a fluorescence microscope with reflected light analysis.

Subsequently, during the deposition of the cells in the hydrophilic zones, their quantification was possible through the analysis of images obtained by fluorescence microscopy. The cell nuclei were stained using DAPI, which reflects a wavelength corresponding to the blue color.

Example 5 - Deposition of fibroblast cells encapsulated in hydrogels

Substrates identical to those in Example 1 were standardized using UVO radiation for 14 minutes. A solution of 1% alginate (mass / volume) was prepared in culture medium with a suspension of fibroblast cells (L929). Solutions of different concentrations (1%, 1.5% and 2% (mass / volume)) in physiological pH collagen buffer solution were prepared separately. The 1% alginate solution was deposited together with one of the collagen solutions in each of the wettable regions at different alginate: collagen ratios (25:75, 50:50, 100: 0) in a dropwise manner, forming a total of 5 μΐ per wettable region. The alginate in each wettable region was crosslinked with a 1 M drop of calcium chloride, whereby hydrogels (semi-interpenetrated networks) were formed with encapsulated cells. Culture medium was added to each region. After 24 hours and 48 hours of cell culture, the cells were evaluated for viability using calcein AM, and in their proliferation using DAPI. The imaging was performed under a fluorescence microscope and the quantification of cells using a computer program for image analysis. 35

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Lisbon, 6 December 2011. 37

Claims (15)

Process for obtaining a surface comprising areas with differences in surface tension, characterized in that it comprises the following steps: a) Providing a flat substrate with a high degree of water repellency, b) Applying a mask to the surface of said surface substrate, c) Applying to the substrate and mask b) a surface tension modification treatment. Process according to the preceding claim, characterized in that the substrate of a), with high water repellency, comprises surfaces whose angles of contact with water are greater than 110 °, more particularly 150 °. A process according to claim 1, wherein the surface tension modifying treatment applied to the substrate b) comprises ultraviolet irradiation with ozone or plasma. Surface, comprising areas with surface tension differences, characterized in that it is obtained by the process described according to any of claims 1 to 3. Surface, according to the preceding claim, characterized in that it comprises water repellent areas and hydrophilic or superhydrophilic areas. 1 Process for the preparation of an article comprising biological material and a surface as claimed in claim 4 or 5, characterized in that a solution or suspension comprising the biological material and suitable medium is deposited to said biological material, The surface of claim 4 or 5. A process according to the preceding claim characterized in that different solutions or suspensions comprising biological material and suitable medium are deposited on said biological material in different hydrophilic or superhydrophilic areas of the surface described according to claim 4 or 5. Process according to claim 6 or 7, characterized in that the medium suitable for said biological material in the form of a biomaterial in the form of a porous structure is obtained by lyophilization, leaching or cross-linking, or in the form of a hydrogel by cross-linking. Process according to claim 6 or 7, characterized in that deposition of the suitable medium to said biological material in the form of biomaterial is carried out by the layer-on-layer technique. Process according to any one of Claims 6 to 9, characterized in that the deposited biological material is fixed. A process according to any one of claims 6 to 10, characterized in that it comprises coating or impregnating the deposited biomaterial. 2 An article characterized in that it is obtained according to the process described in any one of claims 6 to 11. Use of the article as claimed in claim 12, characterized in that it is applied in characterization analyzes or tests of the biological material and interaction materials contained in said article. Use of the article as claimed in claim 12, characterized in that bioengineering is applied. Use according to claim 14, characterized in that it is applied in the development of implantable biomaterials or the release of active agents. Lisbon, July 18, 2012. 3