Collagen-like protein coated medical devices and coating method thereof

Abstract

The present invention refers to a method for coating a collagen like protein onto a surface of a polymer-scaffold comprising or consisting the following steps: a) plasma-treating the surface of the polymer-scaffold, b) providing a collagen like protein in a solvent, to obtain a collagen like protein-containing solution, c) bringing the plasma treated polymer-scaffold and the collagen like protein containing solution into contact followed by an optionally rinsing, d) removing the solvent and thereby obtaining a collagen like protein coated polymer-scaffold. Furthermore, the present invention refers to an article obtained by the method of the present invention. Moreover, the present invention refers to the use of the article according to the invention for wound healing and bone repair as well as for flask-coating and plate-coating in cell culture.

Description

[0001] Collagen-like protein coated medical devices and coating method thereof

[0002] Field of the invention

[0003] The present invention refers to a method for coating a collagen like protein onto a surface of a polymer- scaffold comprising or consisting the following steps: a) plasma-treating the surface of the polymer-scaffold, b) providing a collagen like protein in a solvent, to obtain a collagen like protein-containing solution, c) bringing the plasma treated polymer-scaffold and the collagen like protein containing solution into contact followed by an optionally rinsing, d) removing the solvent and thereby obtaining a collagen like protein coated polymer- scaffold.

[0004] Furthermore, the present invention refers to an article obtained by the method of the present invention. Moreover, the present invention refers to the use of the article according to the invention for wound healing and bone repair as well as for flask-coating and plate-coating in cell culture.

[0005] Description of the related art

[0006] Surface modification strategies such as coating of biomedical products are widely used to improve the biocompatibility and performance of medical device products. In particular implants manufactured with hydrophobic materials, such as poly-L-caprolactone (PCL) or Polyethylene (PE), require a surface modification to ensure a proper cell adhesion and proliferation and to avoid or minimize implant rejection in the body after implantation.

[0007] The applicability of collagen like protein (CLP) as a coating with high stability is challenging, due to the high water-solubility of this material. By using conventional coating procedures, such as dip-coating, the CLP does not form a homogenous graft on the surface. Therefore, the task to be solved was to enable the coating of medical device products with CLP.

[0008] The inventors of the present invention surprisingly found a specific process which allows coating of CLP on a surface of a polymer-scaffold. The inventor of the present invention found, that an oxygen plasma treatment or an acrylic acid plasma treatment of the surface followed by bringing the plasma treated surface and a CLP-solution in contact results in a CLP coated polymer-scaffold, which has a higher amount of CLP-attachment and an increased density of the CLP-coating compared to CLP-coated scaffolds without plasma-treatment.

[0009] Summary

[0010] In a first aspect the present invention refers to a method for coating a collagen like protein onto a surface of a polymer-scaffold comprising or consisting the following steps: a) plasma-treating the surface of the polymer-scaffold, b) providing a collagen like protein in a solvent, to obtain a collagen like protein-containing solution, c) bringing the plasma treated polymer-scaffold and the collagen like protein containing solution into contact followed by an optionally rinsing, d) removing the solvent and thereby obtaining a collagen like protein coated polymer- scaffold.

[0011] In a second aspect the present invention pertains to an article obtained by the method of the present invention.

[0012] In a third aspect the present invention refers to the use of the article according to the invention for implants for wound healing and bone repair as well as for flask-coating and plate-coating in cell culture.

[0013] These and other aspects, embodiments, features, and advantages of the invention will become apparent to a person skilled in the art through the study of the following detailed description and claims. Any feature from one aspect of the invention can be used in any other aspect of the invention. Furthermore, it will readily be understood that the examples contained herein are intended to describe and illustrate the invention but not to limit the invention and that, in particular, the invention is not limited to these examples.

[0014] Brief description of the drawings

[0015] Fig. 1 shows the cell morphology evaluation of MG-63 osteoblast-like cells on Fig. 1A) CLP coated films without pre-treatment; and, CLP coated films after pre-treatment with Fig. 1 B) oxygen plasma activation and Fig. 1C) plasma acryl acid polymerization.

[0016] Detailed description

[0017] Numerical ranges that are indicated in the format “from x to y” also include the stated values. If several preferred numerical ranges are indicated in this format, it is self-evident that all ranges that result from the combination of the various endpoints are also included.

[0018] "At least one" means one or more, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more. "At least one", as used herein in relation to any component, refers to the number of chemically different molecules, i.e. to the number of different types of the referenced species, but not to the total number of molecules. For example, "at least one CLP" means that at least one type of molecule falling within the definition for a CLP is used but that also two or more different types of CLP falling within this definition can be present but does not mean that only one or more molecules of one type of CLP are present.

[0019] All percentages given herein in relation to the compositions or formulations relate to wt.-% relative to the total weight of the respective composition, if not explicitly stated otherwise.

[0020] In the following, “collagen like protein” is referred to as “CLP” as well. In the following, “poly-L-caprolactone” is referred to as “PCL” as well.

[0021] As used herein, the term “biodegradable polymer” refers to a polymer or polymers which degrade in vivo, and wherein erosion or breakdown of the polymer or polymers over time occurs concurrently with or subsequent to release of the therapeutic agent. A biodegradable polymer may be a homopolymer, a copolymer, or a polymer comprising more than two different polymeric units.

[0022] The method for coating a collagen like protein onto a surface of a polymer-scaffold comprises or consists the following steps: a) plasma-treating the surface of the polymer-scaffold, b) providing a collagen like protein in a solvent, to obtain a collagen like protein-containing solution, c) bringing the plasma treated polymer-scaffold and the collagen like protein containing solution into contact followed by an optionally rinsing, d) removing the solvent and thereby obtaining a collagen like protein coated polymer- scaffold.

[0023] In the method at least one collagen-like protein is used. In general, all collagen-like proteins are suitable.

[0024] In a preferred embodiment of the present invention the collagen-like protein is a collagen-like protein from Streptococcus pyogenes, which is preferably the Scl2 protein from Streptococcus pyogenes.

[0025] Expression of collagen-like proteins have been attempted in several systems, including Escherichia coli and Saccharomyces cerevisiae. In one embodiment the at least one collagen-like protein is a bacterial collagen-like protein, preferably produced by fermentation in Pichia, Brevibacillus, Bacillus, Escherichia or Corynebacterium, preferably Pichia pastoris, Brevibacillus choshinensis or Corynebacterium glutamicum.

[0026] In a preferred embodiment the collagen-like proteins may be expressed in Corynebacterium, preferably in Corynebacterium glutamicum.

[0027] One particularly suitable collagen-like protein is derivable from following polynucleotide.

[0028] A polynucleotide encoding an amino acid sequence that is at least > 60%, identical to the amino acid sequence of SEQ ID NO:1 , wherein the polynucleotide is a replicable polynucleotide encoding a collagen- like protein and wherein the amino acid sequence comprises a deletion of at least 38 amino acids at the N- terminus of the amino acid sequence of SEQ ID NO:1 .

[0029] It is preferred, when the amino acid sequence comprises a deletion of between 38 and 74 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:1. This includes a complete deletion of the N- terminal V-domain (comprising 74 amino acids) and different truncations of the V-domain of at least 38 amino acids. In a preferred embodiment, the amino acid sequence that is at least > 60%, identical to the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0030] In a further configuration, the amino acid sequence that is at least > 65%, or > 70%, or > 75%, or > 80%, or

[0031] > 85% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0032] In a preferred configuration, the polynucleotide encodes an amino acid sequence that is at least > 90%,

[0033] > 92%, > 94%, > 96%, > 97%, > 98%, > 99% or 100%, preferably > 97%, particularly preferably > 98%, very particularly preferably > 99%, and extremely preferably 100%, identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0034] In a preferred embodiment of the present invention the polynucleotide is a replicable nucleotide sequence encoding the collagen-like protein from Streptococcus pyogenes.

[0035] Polynucleotide and nucleic acid molecules comprising such sequences and encoding polypeptide variants of SEQ ID NO:1 to 4, which contain one or more insertion(s) or deletion(s) are suitable as well. Preferably, the polypeptide contains a maximum of 5, a maximum of 4, a maximum of 3, or a maximum of 2, insertions or deletions of amino acids.

[0036] Mixture of polypeptides comprising one of the polypeptide variants of SEQ ID NO:1 to 4 and on or more of the truncated variants of the collagen-like protein of SEQ ID NO:5 to 9 can be used as well.

[0037] Plasmids and vectors that comprise the nucleotide sequences according to the invention and optionally replicate in microorganisms of the genera Pichia, Corynebacterium, Pseudomonas or Escherichia are suitable. In a preferred configuration, the vector comprising the nucleotide sequences according to the present invention is suitable for replication in yeast of the genus Pichia pastoris.

[0038] Microorganisms of the genera Pichia, Corynebacterium, Pseudomonas or Escherichia that comprise the polynucleotides, vectors and polypeptides according to the invention are suitable as well. Preferred microorganisms are Pichia pastoris, Brevibacillus choshinensis or Corynebacterium glutamicum.

[0039] Microorganism of the species P. pastoris, E. coli, P. putida or C. glutamicum comprising any of the nucleotide sequences according to the present invention any of the polypeptides or any of the vectors according to the present invention are suitable.

[0040] The microorganism may be a microorganism in which the nucleotide sequence is present in overexpressed form.

[0041] Overexpression according to the invention means, generally, an increase in the intracellular concentration or activity of a ribonucleic acid, a protein (polypeptide) or an enzyme, compared with the starting strain (parent strain) or wild-type strain, if this is the starting strain. A starting strain (parent strain) is taken to mean the strain on which the measure leading to the overexpression was carried out.

[0042] In the overexpression, the methods of recombinant overexpression are preferred. These include all methods in which a microorganism is produced using a DNA molecule provided in vitro. Such DNA molecules comprise, for example, promoters, expression cassettes, genes, alleles, encoding regions etc. These are converted into the desired microorganism by methods of transformation, conjugation, transduction or like methods.

[0043] The extent of the expression or overexpression can be established by measuring the amount of the mRNA transcribed by the gene, by determining the amount of the polypeptide, and by determining the enzyme activity.

[0044] The bacterial collagen-like protein can be obtained in a fermentative process comprising the following steps: a) fermentation of a microorganism according to the present invention in a medium, b) accumulation of the bacterial collagen-like protein in the medium, wherein a fermentation broth is obtained.

[0045] The culture medium or fermentation medium that is to be used must appropriately satisfy the demands of the respective strains. Descriptions of culture media of various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981). The terms culture medium and fermentation medium or medium are mutually exchangeable.

[0046] As carbon source, sugars and carbohydrates can be used, such as, e.g., glucose, sucrose, lactose, fructose, maltose, molasses, sucrose-containing solutions from beet sugar or sugar cane processing, starch, starch hydrolysate and cellulose, oils and fats, such as, for example, soybean oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols such as, for example, glycerol, methanol and ethanol, and organic acids, such as, for example, acetic acid or lactic acid.

[0047] As nitrogen source, organic nitrogen compounds such as peptones, yeast extract, meat extract, malt extract, corn-steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used. The nitrogen sources can be used individually or as a mixture.

[0048] As phosphorus source, phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used.

[0049] The culture medium must, in addition, contain salts, for example in the form of chlorides or sulphates of metals such as, for example, sodium, potassium, magnesium, calcium and iron, such as, for example, magnesium sulphate or iron sulphate, which are necessary for growth. Finally, essential growth substances such as amino acids, for example homoserine and vitamins, for example thiamine, biotin or pantothenic acid, can be used in addition to the above-mentioned substances.

[0050] Said starting materials can be added to the culture in the form of a single batch or supplied in a suitable manner during the culturing.

[0051] Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acid compounds such as phosphoric acid or sulphuric acid, are used in a suitable manner for pH control of the culture. The pH is generally adjusted to 6.0 to 8.5, preferably 6.5 to 8. For control of foam development, antifoams can be used, such as, for example, polyglycol esters of fatty acids. For maintaining the stability of plasmids, suitable selectively acting substances such as, for example, antibiotics, can be added to the medium. The fermentation is preferably carried out under aerobic conditions. In order to maintain said aerobic conditions, oxygen or oxygen-containing gas mixtures such as, for example, air, are introduced into the culture. The use of liquids that are enriched with hydrogen peroxide is likewise possible. Optionally, the fermentation is carried out at superatmospheric pressure, for example at a superatmospheric pressure of 0.03 to 0.2 MPa. The temperature of the culture is usually 20°C to 45°C, and preferably 25°C to 40°C, particularly preferably 30°C to 37°C. In the case of batch or fed-batch processes, the culturing is preferably continued until an amount sufficient for the measure of obtaining the desired organic chemical compound has formed. This goal is usually reached within 10 hours to 160 hours. In continuous processes, longer culture times are possible. Due to the activity of the microorganisms, enrichment (accumulation) of the fine chemicals in the fermentation medium and / or in the cells of the microorganisms occurs.

[0052] Examples of suitable fermentation media may be found, inter alia, in patent documents US 5,770,409, US 5,990,350, US 5,275,940, WO 2007 / 012078, US 5,827,698, WO 2009 / 043803, US 5,756,345 or US 7,138,266; appropriate modifications may optionally be carried out to the requirements of the strains used.

[0053] The process may be characterized by a process which is selected from the group consisting of batch process, fed-batch process, repetitive fed-batch process and continuous process.

[0054] The process may be further characterized by a fine chemical, or a liquid, or a solid fine chemical-containing product is obtained from the fine chemical-containing fermentation broth.

[0055] The performance of the processes or fermentation processes according to the invention with respect to one or more of the parameters selected from the group of concentration (compound formed per volume), yield (compound formed per carbon source consumed), volumetric productivity (compound formed per volume and time) and biomass-specific productivity (compound formed per cell dry mass or bio dry mass and time or compound formed per cell protein and time) or other process parameters and combinations thereof, is increased by at least 0.5%, at least 1 %, at least 1 .5% or at least 2%, based on processes or fermentation processes with microorganisms in which the promoter variant according to the invention is present.

[0056] Owing to the measures of the fermentation, a fermentation broth is obtained which contains the desired collagen-like protein, and preferably amino acid or organic acid.

[0057] Then, a product in liquid or solid form that contains the collagen-like protein is provided or produced or obtained.

[0058] A fermentation broth means, in a preferred embodiment, a fermentation medium or nutrient medium in which a microorganism was cultured for a certain time and at a certain temperature. The fermentation medium, or the media used during the fermentation, contains / contain all substances or components that ensure production of the desired collagen-like protein and typically ensure growth and / or viability.

[0059] On completion of the fermentation, the resultant fermentation broth accordingly contains a) the biomass (cell mass) of the microorganism resulting from growth of the cells of the microorganism, b) the desired collagen-like protein formed in the course of the fermentation, c) the organic by-products possibly formed in the course of the fermentation, and d) the components of the fermentation medium used, or of the starting materials, that are not consumed by the fermentation, such as, for example, vitamins such as biotin, or salts such as magnesium sulphate.

[0060] The organic by-products include substances which are generated in addition to the respective desired compound by the microorganisms used in the fermentation and are possibly secreted.

[0061] The fermentation broth is withdrawn from the culture vessel or the fermentation container, optionally collected, and used for providing a product in liquid or solid form containing the collagen-like protein. The expression "obtaining the collagen-like protein-containing product" is also used therefor. In the simplest case, the collagen-like protein-containing fermentation broth withdrawn from the fermentation container is itself the product obtained.

[0062] By way of one or more of the measures selected from the group a) partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%) removal of the water, b) partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%) removal of the biomass, wherein this is optionally inactivated before the removal, c) partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, > 99.3%, > 99.7%) removal of the organic by-products formed in the course of the fermentation, and d) partial (> 0%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, > 99.3%, > 99.7%) removal of the components of the fermentation medium used orthe starting materials that are not consumed by the fermentation, a concentration or purification of the desired collagen-like protein is achieved from the fermentation broth. In this manner, products are isolated that have a desired content of the compound.

[0063] The partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80% to < 100%) removal of the water (measure a)) is also termed drying.

[0064] In a variant of the process, by complete or virtually complete removal of the water, the biomass, the organic by-products and the non-consumed components of the fermentation medium used, pure (> 80% by weight, > 90% by weight) or high-purity (> 95% by weight, > 97% by weight, > 99% by weight) product forms of the desired collagen-like protein, preferably bacterial collagen-like protein, are successfully arrived at. For the measures according to a), b), c) or d), a great variety of technical instructions are available in the prior art.

[0065] In the case of processes for producing bacterial collagen-like protein processes are preferred in which products are obtained that do not contain any components of the fermentation broth. These products are used, in particular, in human medicine, in the pharmaceuticals industry, and in the food industry.

[0066] In an embodiment the solvent of the CLP containing solution is an aqueous medium, preferably a buffer, more preferably 2-[4-(2-Hydroxyethyl)-piperazin-1-yl]ethane-1 -sulfonic acid (HEPES) or phosphate buffered saline (PBS) or mixtures thereof, most preferably water. In an embodiment the CLP containing solution has a pH value of 4 to 8, preferably 4 to 7, more preferably 4 to 5, most preferably 4 measured with pH-strips and a concentration of 1 to 10 wt%, preferably 3 to 8 wt%, more preferably 5 to 7 wt%, most preferably 6 wt%.

[0067] The present invention requires a polymer scaffold, which is coated with a CLP.

[0068] In an embodiment the polymer scaffold is selected from granulates or films, preferably selected from films.

[0069] In an embodiment the polymer is selected from a poly (lactide), a poly(glycolide), a poly(lactide-co- glycolide), a poly(caprolactone), a poly (orthoester), a poly(phosphazene), a poly(hydroxybutyrate) a copolymer containing a poly(hydroxybutarate), a poly(lactide-co-caprolactone), a polycarbonate, a polyesteramide, a polyanhydride, a poly(dioxanone), a poly(alkylene alkylate), a copolymer of polyethylene glycol and a polyorthoester, a polyurethane, a poly(amino acid), a polyamide, a polyesteramide, a polyetherester, a polyacetal, a polycyanoacrylate, a poly(oxyethylene) / poly(oxypropylene) copolymer, polyacetals, polyketals, polyphosphoesters, polyhydroxyvalerates or a copolymer containing a polyhydroxyvalerate, polyalkylene oxalates, polyalkylene succinates, poly(maleic acid) or mixtures thereof, preferably selected from poly-L- caprolactone, polyethylene or mixtures thereof.

[0070] In an embodiment the poly-L-caprolactone polymer has a weight average molecular weight of 5000 to 140000 g / mol, measured via GPC using polystyrene standards.

[0071] In an embodiment the poly-L-caprolactone polymer as an inherent viscosity of 0.1 to 2.5 dL / g, preferably 0.5 to 2.5 dL / g or 0.8 to 2.0 dL / g in CHCh at 25°C measured with a Ubbelhode size 0c glass capillary viscometer.

[0072] In an embodiment the poly-L-caprolactone polymer has a melting temperature of 50 to 70°C, preferably 55 to 65°C.

[0073] In an embodiment the polymer is a biodegradable polymer.

[0074] In an embodiment the polymer-scaffold can additionally comprise an additive.

[0075] In an embodiment the amount of additive is 1 wt% to 20 wt%, preferably 1 wt% to 10 wt%, more preferably 2 wt% to 5 wt%, most preferably 3.8 wt%, based on the total weight of the polymer-scaffold, respectively.

[0076] In an embodiment the additive has a particle size of at most 200 pm, preferably at most 100 pm, determined via laser diffraction. In an embodiment the additive is a mineral, preferably a phosphate mineral, more preferably an apatite, most preferably hydroxyapatite.

[0077] The hydroxyapatite can be a single type of hydroxyapatite or a mixture of different hydroxyapatites, for example sintered and unsintered hydroxyapatites.

[0078] In an embodiment the hydroxyapatite is generated through a milling process such as jet milling, impact milling, centrifugal milling, pin milling, knife milling, cryomilling, mix milling, impact milling, air classifier milling, and hybrid methods of the same. In another embodiment, the polymer-based material is blended with the hydroxyapatite using but not limited to high shear mixing, inversion blending, solvent blending, and hybrid methods of the same.

[0079] In an embodiment the plasma-treatment is performed via an oxygen-plasma comprising an oxygen-flux of 30 to 250 seem, preferably 50 to 200 seem, more preferably 75 to 150 seem, most preferably 120 seem, and a plasma power of 25 to 60 W, preferably 30 to 60 W, more preferably 35 to 60 W, most preferably 60 W for 20 to 300 seconds, preferably 60 to 240 seconds, more preferably 120 to 200 seconds, most preferably 180 seconds and a pressure of 1 ■ 104mbar to T 101mbar, preferably T 103mbar to T 102mbar, more preferably 2-103mbar to 8-10'3mbar, most preferably 4-103mbar.

[0080] In an embodiment the plasma-treatment is performed via a plasma polymerized acrylic acid. For this reason the polymer-scaffold is pre-treated using an argon-plasma with an argon flux of 30 to 250 seem, preferably 50 to 200 seem, more preferably 75 to 150 seem, most preferably 120 seem, and a plasma power of 25 to 60 W, preferably 30 to 60 W, more preferably 35 to 60 W, most preferably 60 W for 20 to 300 seconds, preferably 60 to 240 seconds, more preferably 120 to 200 seconds, most preferably 180 seconds and a pressure of T 104mbar to T 101mbar, preferably T 103mbar to T 102mbar, more preferably 2-103mbar to 8- 10'3mbar, most preferably 4-103mbar. After the pre-treatment the plasma deposition of polymerized acrylic acid is performed with an argon flux of 30 to 250 seem, preferably 50 to 200 seem, more preferably 75 to 150 seem, most preferably 120 seem, an acrylic acid flux with a pressure of 1 -104mbar to T101mbar, preferably T103mbar to T102mbar, more preferably 2-103mbar to 8-103mbar, most preferably 7.5- 10-3mbar and a plasma power of 25 to 60 W, preferably 30 to 60 W, more preferably 35 to 55 W, most preferably 50 W for 20 to 300 seconds, preferably 60 to 300 seconds, more preferably 120 to 300 seconds, most preferably 240 seconds.

[0081] In an embodiment the plasma treated polymer scaffolds are brought into contact with the CLP containing solution, preferably doused with the CLP containing solution, more preferably submerged in the CLP containing solution preferably for 0.5 to 60 minutes, more preferably 1 to 30 minutes or more preferably 1 to 15 minutes, most preferably for 5 minutes.

[0082] In an embodiment the plasma treated polymer scaffold which is doused with the CLP containing solution or submerged in the CLP containing solution is optionally rinsed with an aqueous medium, preferably a buffer, more preferably 2-[4-(2-Hydroxyethyl)-piperazin-1-yl]ethane-1 -sulfonic acid (HEPES) or phosphate buffered saline (PBS) or mixtures thereof, most preferably water. In an embodiment the solvent of the optionally rinsed CLP-polymer scaffold is removed, preferably the optionally rinsed CLP-polymer scaffold is dried, preferably air-dried, more preferably compressed-air dried.

[0083] In an embodiment the solvent of the rinsed CLP-polymer scaffold is air-dried using compressed air for 10 seconds to 1 minute, preferably 10 seconds.

[0084] Furthermore the present invention provides an article which is obtained by the method of the present invention.

[0085] In an embodiment the article is a medical device, preferably selected from implants, screws, meshes and ligaments..

[0086] In an embodiment the article has a water contact angle (WCA) of > 20°, preferably 5° to 20°, more preferably 10° to 20°, most preferably 16° to 20°

[0087] The measurement of WCA was used to determine wettability and hydrophilicity of the CLP coated polymer scaffolds. The WCA is related to the hydrophilicity of the surface. The lower the WCA value the higher is the hydrophilicity of the surface. WCA values between 30° to 90° indicate a low hydrophilicity, values below 20° indicate hydrophilic surfaces. The WCA was measured according to the drop shape method using the Drop Shape Analyzer - Kliss Model G-23.

[0088] In an embodiment the article of the present invention has a CLP-coating density of 250 pg / cm2to 600 pg / cm2, preferably 260 pg / cm2to 550 pg / cm2, more preferably 280 pg / cm2to 450 pg / cm2, most preferably 289 pg / cm2to 346 pg / cm2.

[0089] The CLP-coating density is determined by quantifying the total protein content, after detaching the CLP from the implant surface. The leaching procedure is done by incubating the specimen at 60°C, during 30 min within the two component buffer used during the Pierce BCA protein assay (Carbonate Buffer and Cupper Buffer). The complete detachment of the protein is confirmed by means of XPS measurement. The total amount of CLP is quantified and referred to the whole surface of the coated implant, leading finally to the CLP density.

[0090] In an embodiment the article according to the invention has an increased stability of the CLP-coating on a polymer scaffold compared to a CLP coated polymer scaffold without plasma-treatment.

[0091] In an embodiment, after seeding cells on the surface of the article according to the invention, the article shows an increased homogeneous distribution of cells in comparison to a CLP coated polymer scaffold without plasma-treatment.

[0092] A proper and homogenous interaction of cells with the surface of implants is crucial for the performance of medical devices in regenerative medicine. For this purpose, 1 mL of a suspension with 1x 106cells / mL MG-63 osteoblasts-like cells were seeded on the surfaces of the article according to the invention and cultivated in media for 24 h. Finally, cells were stained with a Alexa 555 / Phalloidin dye, and visualized under the microscope. In addition the present invention provides the use of the article according to the invention for wound healing and bone repair, preferably bone repair, cartilage repair, skin repair, sutures or neo-ligaments, as well as for cell culture applications, preferably CLP coated cell culture flasks and plates.

[0093] Examples

[0094] Example 1 : Procedure for coating PCL films

[0095] In a first step, the PCL film was treated using an oxygen plasma technology. The plasma treatment was performed with an oxygen flux of 120 seem and a plasma power of 60 W for 3 min. The base pressure was set to 5 10'4mbar, the process pressure was 4-103mbar. The plasma treatment was also performed via a plasma polymerized acrylic acid. Therefore the PCL film was pre-treated using an argon plasma (flux = 120 seem, 60 W, 3 min, 4-10-3mbar). The base pressure was set to 5 10-4mbar. The plasma deposition of pAA was performed with an argon flux of 120 ccm and the AA flux was adjusted to a process pressure of 7.5- 10-3mbar. The plasma power was 60 W. After 4 min deposition time the coating thickness was approximately 38 nm.

[0096] Before the plasma-treatment or in the meanwhile or afterwards, the CLP was dissolved in water at a final concentration of 6% w / v using an ultrasonic bath for a duration of 10 min. The final pH was 4.

[0097] Then the PCL film was submerged in the CLP solution for 5 min, rinsed with water and finally dried using compressed air.

[0098] Example 2: Water contact angle

[0099] The WCA was determined on untreated PCL-films and after plasma-treatment and coating to determine the degree of surface modification.

[0100] Untreated and CLP coated PCL-films showed a slight decrease of the WCA (from 64±1 ° to 48±2°) indicating a slight increase of hydrophilicity and therefore a small amount of CLP attachment. On the other hand plasma treated PCL-films without CLP-coating showed a significant decrease of the WCA (41 ±1 ° resp. 37± 1 °). Those values decreased even more after the CLP coating (17±1 ° resp. 18±2°). The study reveals that increasing the wettability of the surface using plasma treatments, the immobilization of CLP can be improved considerably in comparison to untreated polymer scaffolds. Example 3: Evaluation of the cell morphology

[0101] The coated PCL films were submitted to a cell morphology test to evaluate the adhesion and fitness of the cells on the surfaces. For comparison purposes CLP coated PCL films without plasma-treatment and CLP coated PCL-films after plasma-treatment were included in the study. As shown in figure 1 , untreated surfaces showed a very inhomogeneous distribution of the cells, indicating unproper and inefficient coating with CLP. On the other hand, plasma treated surfaces showed an improved distribution and grafting of the cells. Example 4: Density of CLP-coatinq

[0102] The density of the CLP-coating was measured of untreated CLP-coated PCL-films and of plasma-treated CLP-coated PCL-films. While the untreated CLP-coated PCL-film showed a CLP-coating density of 184.07 pg / cm2, the density of the CLP-coating of plasma treated PCL-films clearly increased (289.45 pg / cm2resp. 346.83 pg / cm2). These results indicate that the plasma-treatment according to the invention has a positive effect on the density of the CLP-coating.

Claims

Claims1 . Method for coating a collagen like protein onto a surface of a polymer-scaffold comprising or consisting the following steps: a) plasma-treating the surface of the polymer-scaffold, b) providing a collagen like protein in a solvent, to obtain a collagen like protein-containing solution, c) bringing the plasma treated polymer-scaffold and the collagen like protein containing solution into contact followed by an optionally rinsing, d) removing the solvent and thereby obtaining a collagen like protein coated polymer- scaffold.

2. Method according to claim 1 , wherein in step a) the plasma-treatment is performed via an oxygen-plasma with an oxygen flux of 30 to 250 seem, a plasma power of 25 to 60 W for 20 to 300 seconds and a pressure of 1 ■ 10-4to 1 ■ 10-1mbar.

3. Method according to any of the preceding claims, wherein the plasma-treatment of step a) comprises a pre-treatment step of the polymer-scaffold by an argon plasma with an argon flux of 30 to 250 seem, a plasma power of 25 to 60 W for 20 to 300 seconds and a pressure of 1 ■ 10-4to 1 -10’1mbar and, after the pre-treatment step the plasma deposition of the polyacrylic acid is performed with an argon flux of 30 to 250 seem, an acrylic acid flux with a pressure of 1 ■ 10 '4to1 ■ 10"1mbar and a plasma power of 25 to 60 W for 20 to 300 seconds.

4. Method according to any of the preceding claims, wherein in step b) the collagen-like-protein containing solution has a pH-value of 4 to 8 and a concentration of 1 to 10% .

5. Method according to any of the preceding claims, wherein in step b) the solvent is an aqueous solvent.

6. Method according to any of the preceding claims, wherein in step c) c1) the plasma treated polymer-scaffold is submerged in the collagen-like-protein containing solution for 30 seconds to 1 hour and c2) subsequently the collagen-like-protein coated polymer-scaffold is optionally rinsed with water.

7. Method according to any of the preceding claims, wherein in step d) the collagen-like-protein coated polymer-scaffold is air-dried for 10 seconds to 1 minute.

8. Method according to any of the preceding claims, wherein the polymer-scaffold is selected from films or granulates.

9. Method according to any of the preceding claims, wherein the polymer is selected from a poly(lactide), a poly(glycolide), a poly(lactide-co-glycolide), a poly(caprolactone), a poly(orthoester), a poly(phosphazene), a poly(hydroxybutyrate) a copolymer containing a poly(hydroxybutarate), a poly(lactide-co-caprolactone), a polycarbonate, a polyesteramide, a polyanhydride, a poly(dioxanone), a poly(alkylene alkylate), a copolymer of polyethylene glycol and a polyorthoester, a polyurethane, a poly(amino acid), a polyamide, a polyesteramide, a polyetherester, a polyacetal, a polycyanoacrylate, a poly(oxyethylene) / poly(oxypropylene) copolymer, polyacetals, polyketals, polyphosphoesters, polyhydroxyvalerates or a copolymer containing a polyhydroxyvalerate, polyalkylene oxalates, polyalkylene succinates, poly(maleic acid), poly-L-caprolactone, polyethylene or mixtures thereof, wherein the polymer-scaffold can additionally comprise hydroxyapatite in an amount of 1 wt% to 20 wt%, based on the total weight of the polymer-scaffold.

10. Method according to any of the preceding claims, wherein the collagen-like-protein is obtained from pichia pastorius or Corynebacterium.11 . Method according to any of the preceding claims, wherein the collagen-like-protein is derivable from a polynucleotide encoding an amino acid sequence that is at least > 60% identical to the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

12. Article obtained by the method of any of the preceding claims, wherein the article is preferably a medical device, more preferably selected from implants, screws, meshes, ligaments.

13. Article according to claim 12, wherein the article has a water contact angle of 20°, measured according to the drop shape method using the Drop Shape Analyzer Kuss Model G-23.

14. Article according to claims 12 or 13, wherein the article has a CLP-coating density of 250 to 600 pg / cm2.

15. Use of the article according to any of claims 12 to 14 for wound healing and bone repair and for flask-coating and plate-coating in cell culture.