Curved or tubular collagen scaffolds

Abstract

A method for producing a curved or tubular collagen scaffold, comprising the steps of: a) providing a substrate; b) cleaning the surface of the substrate; c) functionalizing the surface of the substrate by silanization using a silane or by coating with a metal or carbon; d) depositing one or more layers of a composition comprising collagen onto the substrate; e) inducing self-assembly by adjusting the pH to form one or more layers of nanofibers comprising collagen; f) inducing coiling by fixing the one or more layers of nanofibers to form a curved or tubular nanofiber protein layer comprising collagen;g) Separating the curved or tubular nanofiber protein layer and the substrate, thereby obtaining the curved tubular collagen scaffold, and wherein step f) comprises a further step in which the fixed nanofiber protein layer comprising collagen is allowed to dry.;

Description

[Technical field] The present invention relates to a method for producing a curved or tubular collagen scaffold, comprising the following steps: a) providing a substrate; b) purifying the surface of the substrate; c) functionalizing the surface of the substrate by silanization using a silane or by coating it with a metal, such as gold, or carbon; d) depositing one or more layers of a composition comprising collagen onto the substrate; e) inducing self-assembly by adjusting the pH to form one or more layers of nanofibers comprising collagen; f) fixing the one or more layers of nanofibers to form a curved or tubular nanofiber protein layer comprising collagen;(g) Separating the curved or tubular nanofiber protein layer and the substrate, thereby obtaining the curved or tubular collagen scaffold, and wherein step (f) comprises a further step in which the fixed nanofiber protein layer comprising collagen is allowed to dry; the curved or tubular collagen scaffold and its use, in particular for tissue engineering. [State of the art] The human body is a dynamic system in which each organ comprises various cell types embedded in extracellular matrices (ECM), which consist primarily of proteins. In less complex organs, the transport of substances to and from the organ occurs via passive diffusion. However, complex transport involving powerful muscle contractions is mediated by tubular tissues, which are composed of specialized cells depending on their location and the function they are to perform. Therefore, the entire human body and its organ systems contain tubular tissue structures, such as the vascular system (arteries, veins, capillaries), gastrointestinal components (esophagus, trachea), urinary tract components (ureters, urethra, bladder), and the nervous system. Like most organ systems, tubular tissue is susceptible to disease and dysfunction.This also necessitates suitable regeneration strategies, such as the use of donor tissue or an autologous implant, or the replacement of lost tubular tissue by creating scaffolds with a similar architecture for implantation into the body. The main disadvantage of donor tissue or autografts is the limited availability of either the patient's own or donor tissue, combined with the risk of rejection by the patient's immune response. Tubular implants made of synthetic materials, on the other hand, are readily available, for example, as vascular grafts and stents for use in cardiac surgery or as nerve conduits to support nerve regeneration. Nevertheless, the production of tubular constructs from natural polymers that support cell growth through biochemical and topographical cues remains the focus of current research and constitutes the fundamental task of this disclosure of invention. Typical materials used in cardiac therapy are metals and synthetic polymers, while nerve fibers are often made from synthetic polymers. When such synthetic materials are used to replace tubular tissue, they often fail to meet demanding anatomical and mechanical specifications, do not support cell adhesion through biochemical cues, and often have long-term patent concerns that could lead to occlusion and dysfunction of the tubular scaffold. Furthermore, stent technology has a number of disadvantages, such as potential inflammatory reactions, calcification, stent displacement, and discomfort or discrepancies in mechanical properties compared to the surrounding tissue. Stent technology has also been widely implemented in the treatment of tracheal diseases, for example, using metal stents, ultraflex stents, or hybrid stents made of reinforced silicone with metal rings. Tissue engineering techniques have also reported successful tracheal regeneration using several types of synthetic materials, decellularized tissues, and biodegradable polymers. Several natural polymers, such as hyaluronan, collagen, and silk fibroin, have also been used for tracheal tissue engineering. Blood vessel replacement strategies include endothelial cell-seeded synthetic grafts, collagen-based blood vessel analogs, decellularized tissues, and biodegradable synthetic blood vessels. Early blood vessel replacements were made from synthetic polymers such as Dacron, Gore-Tex, or expanded polytetrafluoroethylene (ePTFE) and proved clinically efficient in replacing large-diameter vessels (≥ 6 mm). Thrombosis has been reported in smaller vessels, along with reduced patentness. Artegraft, Procol, and Cryovein became the first widely available acellular tissue-engineered vascular grafts (TEVGS) in the late 1970s.The first collagen hydrogel tube structure was developed by Weinberg and Bell, but had limited success due to poor mechanical properties, which were improved by Tranquillo and colleagues through the development of oriented collagen fibrils using magnetic pre-alignment during the collagen fibrillation process. In nerve regeneration, FDA-approved nerve conduits (NGCs) are available. However, these tubular NGCs provide an insufficient cross-sectional area and are unable to successfully repair critically large nerve gaps in the human body. The basic design of these tubes can be similar, and they are primarily manufactured from synthetic polymers such as PGA and PLC or hydrogel, as well as natural polymers like collagen, using various manufacturing techniques, such as mesh rolling, precipitation, or dip coating on a rotating mandrel. Electrospun PLGA fibers have also been investigated as novel spiral scaffolds for improved cellular activity and tissue formation in nerve repair. The complexity of tubular tissue in the gastrointestinal tract, along with its functional role, has created the need to develop artificial intestines based on biodegradable and mechanically stable, yet flexible, scaffolds. To date, there are very limited studies on the implantation of a fully tissue-engineered tubular esophagus. For the regeneration of tubular tissue in the urinary tract, decellularized tissue matrices and molded collagen-based scaffolds have been investigated. Despite these advances, there are currently no long-term, sustainable replacement strategies for tubular tissue in the gastrointestinal or urinary tract, so donor tissue transplantation remains the only viable solution. When designing scaffolds to replace tubular tissue, a load-bearing framework for cells must be provided without sacrificing the overall function of the resulting structure. Furthermore, there is often a need to construct an intact lumen capable of supporting the construction of an epithelium and other materials. To date, scaffolds for tubular tissue regeneration have been fabricated using a variety of techniques, including casting, electrospinning, rolling, 3D printing, and decellularization. All of these techniques rely on extensive, high-end instrumentation and require significant time to produce tubular structures. Electrospinning: Electrospinning involves the solubilization of a natural or synthetic polymer in an organic solvent, which is then expelled from a syringe into an electric field. The surface tension / viscosity of the liquid and the strength of the electric field are important factors during electrospinning. For tubular scaffolds, the liquid is accelerated in the electric fields to form a neutral or oppositely charged rotating mandrel. Once the solvent evaporates, a tubular nanofiber network is created. However, this technique suffers from disadvantages such as the use of organic solvents, high-energy electric fields, and high concentrations of the (bio)polymer required for scaffold fabrication, which can impair protein functionality for subsequent cell culture. 3D bioprinting: 3D bioprinting emerged as a technology capable of producing complex structures along with cells and providing a support matrix for constructing tubular organ structures. Three types of 3D bioprinting are available: extrusion bioprinting, droplet bioprinting, and laser bioprinting. More specifically, to create tubular tissues via concentric ring arrangements, vascular networks using volatile inks, or Kenzan printing of cell spheroids on needles, coaxial tube formation from a modified nozzle and coaxial extrusion onto rotating glass rods have been implemented. Although 3D bioprinting has emerged as a high-precision method for creating scaffolds, this technique is not cost-effective and also requires specialized equipment and skilled personnel for its operation.This also means that a considerable amount of time is required to produce individual tubular scaffolds in a multi-step, sequential process. Furthermore, bioprinting is limited to suitable bioinks that can sustain the printing process and also support cell growth. Decellularization: The construction of a scaffold from native tissue is the main focus of this technique. It can be applied to the decellularization of entire organs or to the decellularization of tissue / specific structures, both of which are usually accomplished by removing the native tissue and then treating it to completely remove cells (via enzymatic or detergent-based lysis). The tissue is then washed and lyophilized to be used as a scaffold for tissue engineering. This technique is quite biocompatible and has minimal chances of immune rejection when tissues are obtained from a suitable donor. However, tissues from different sources induce significant variations in scaffold production and may—depending on the source—carry certain biomolecular factors that can impair subsequent cell growth.In particular, native tissue sources may have limited availability and may be immunogenic depending on the host tissue. Furthermore, the processing of ex vivo samples is complex and requires specific biological standards, i.e., S1 / S2 laboratory facilities, and sterilization equipment. Finally, decellularized scaffolds may contain biochemical entities that could have detrimental effects on cell growth in the cultured tissue. Casting: Casting is one of the most widely used manufacturing methods in tissue engineering. In this approach, a liquid is poured or injected into a container / mold, causing it to solidify through various processes such as gelation, crosslinking, and solvent evaporation. This technique can be applied to a wide range of materials and is very easy to use. However, it is difficult to produce scaffolds with a tubular architecture that have an internal cavity, as the solidified scaffold often cannot be separated from the mold material. Consequently, only certain geometries could be achieved depending on the mold dimensions; more complex structures cannot be built. Furthermore, homogeneous cell seeding into the lumen cavity of cast tubular scaffolds is difficult to achieve, and simultaneous rotation may be necessary. Dip coating: A simplified, efficient method for tubular tissue construction involves using a rod that can be alternately dipped into a cell-loaded hydrogel and a crosslinking agent using repeated dips in a multilayer tubular construct. A degree of control over layer thickness could be achieved by introducing motors to rotate and dip the rods. However, this is a repetitive, multi-step process and therefore quite time-consuming for realistic applications in tubular scaffold fabrication. Rolling: The rolling technology is based on a flat substrate—either polymer sheets or cell-derived ECM—which is rolled into a tube manually or using an automated setup, usually assisted by a mandrel. Rolling is the only fabrication method most specifically suited for producing tubular scaffolds from natural polymers such as collagen or elastin, as well as from synthetic polymers, and as such has had a significant impact on tubular tissue engineering. However, specialized equipment is required for rolling tubular constructs, which is a time-consuming, multi-step technique. Rolled structures can be produced by stretching techniques, such as those used to roll up inorganic membranes made of various materials, including different metals, e.g., Pt and Pd / Fe / Pd, or ceramics, e.g., TiO2, ZnO, Al2O3.However, when stretching techniques are used in combination with rolling, it has not yet been possible to process natural polymers into tubular scaffolds—only ceramics or metals could be processed in this way. In other studies, acellular collagen and elastin films were rolled into a tube using a stainless steel mandrel to create an acellular version of native arterial tissue. Tubular structures with layered cell strata using a self-assembly mechanism based on specific properties of the underlying substrate have been developed. Generally, in these studies, a polymer film was produced and sewn into a tubular form. Self-assembly mechanisms or other rolling strategies that can be performed under sterile conditions have the main advantage of allowing cell seeding prior to rolling.In these studies, rolling was initiated using either mechanically tensioned films or shape-memory polymers. However, sealing free edges of a rolled-up construct is difficult once it is seeded with cells. Thermoresponsive polymers in combination with automated rolling were used to produce tubular cell scaffolds. A thermoresponsive coating of poly(N-isopropylacrylamide)-grafted gelatin or poly(glycidyl ether) brushes was applied to a rotating mandrel, facilitating cell sheet attachment, subsequent peeling, and rolling. This approach allows for the production and gentle harvesting of confluent cell sheets with their extracellular matrix.Furthermore, the arrangement of cell sheets into tubular structures was achieved through cell self-assembly to produce a fully biological vascular graft with excellent mechanical properties, but this had disadvantages during translation in clinics. ECM proteins such as collagen play a crucial role in the structural and functional stability of tubular tissues, with collagen being one of the most abundant proteins, and are involved in many cellular processes, including tissue repair. Unlike any other synthetic material, collagen can provide vital binding sites to facilitate cell adhesion, migration, and proliferation. For example, the repair of peripheral nerves using tubular collagen-based conduits has been reported, along with the rolling of collagen into tubular and hollow constructs for fibroblast cell growth and urinary tissue engineering applications. Brown and coworkers used a plastic compression technique to fabricate tubular collagen constructs with enhanced mechanical properties. Nevertheless, all the discussed techniques share the disadvantage of lacking biochemical cues, as many existing approaches to creating tubular tissue replacements involve the use of synthetic materials that are not part of native tissue. Therefore, current tubular constructs often fail to mimic the biochemical cues of the native ECM to facilitate cell adhesion, migration, and proliferation. Furthermore, they lack porosity. In the native ECM, collagen is the primary component, forming a highly porous, fibrous network into which cells can migrate. Besides biochemical cues, ECM porosity and topography play a significant role in cell attachment, even in tissues with a tubular architecture.However, for tubular constructs fabricated using the techniques mentioned above, it is often difficult to create scaffolds with a nanoporous or fibrous architecture that mimics the topographical features of the native ECM. Ultimately, the compositions are quite limited. Besides collagen, other proteins such as fibronectin or elastin and polysaccharides such as hyaluronan are also components of the ECM. However, no tubular constructs composed of multiple biopolymers have yet been fabricated using the techniques described above. Another approach is the tissue engineering technique of tubular cell assemblies through self-assembly without the use of scaffolds, known as TESA. This method relies on natural cell-cell interactions, in which cells deposit their own ECM into the tubular cell spheroid. This approach to developing tubular tissues does not require high-end instrumentation. So far, TESA has been used to generate entirely biological, unprocessed, collagen-rich tissues. However, the TESA technique does not provide a scaffold to support cell growth; that is, cells cannot be pre-seeded onto the tubular scaffold to promote tissue repair.Therefore, this approach would require large quantities of healthy donor cells (as in autografts) combined with long cultivation times outside the body under in-vitro conditions to enable sufficient ECM production before the tubular cell scaffold can actually be reimplanted into the defect site. During this week-long procedure, the defect site would remain untreated, significantly delaying the overall tissue repair process. Finally, methods based on synthetic / inorganic polymers were developed for the simultaneous coiling of nanotubes for catalytic applications and drug delivery. However, due to the materials involved in this self-assembly process, such tubular constructs do not meet the criteria to mimic the native ECM nanotopography and biochemical composition, as required for tissue engineering applications. It is an object of the present invention to provide a method for the production of tubular collagen scaffolds that overcomes the numerous disadvantages of the prior art. The following methods are known from the prior art: Dutta et al. (“Effect of Collagen Nanofibers and Silanization on the Interaction of HaCaT Keratinocytes and 3T3 Fibroblasts with Alumina Nanopores”, Applied Bio Materials, 2021, Vol. 4, pp. 1852-1862) investigate the surface functionalization of nanoporous alumina membranes by silanization with (3-aminopropyl)triethoxysilane (APTES) and by coating with collagen nanofibers. The modification of the material surface to influence the interaction with 3T3 fibroblasts and HaCaT keratinocytes is examined. This publication describes in particular the deposition of collagen nanofibers on an aluminum oxide substrate. Specifically, the publication discloses that, in a first step, aluminum oxide membranes are prepared. These are then purified with a Piranha solution. Subsequently, the purified substrate is silanized.The following describes how collagen nanofibers are arranged on the AAO membrane. These arranged fibers are then exposed to a PBS buffer solution with a pH of 7.4. Finally, the resulting product is cross-linked using a glutaraldehyde solution. US 2018 / 0250437 A1 discloses a process for producing a three-dimensional polymer scaffold (3D-PS) for applications in tissue engineering, the process comprising the following steps: providing a polymeric precursor scaffold (p-PS) containing at least one biodegradable natural polymer (PA); treating the polymeric precursor scaffold (p-PS) with a crosslinking agent comprising glutaraldehyde to induce crosslinking of the natural polymer (PA), thereby obtaining a crosslinked polymer scaffold (x-PS); subsequently subjecting the crosslinked polymer scaffold (x-PS) to a low-pressure plasma treatment under exposure to an ionized, oxygen-containing gas plasma at a pressure in the range of 10⁻³ to 10⁻⁶ bar and a temperature below 40 °C. US 2021 / 0213162 A1 discloses a process for producing fibrous fibrinogen biomaterials that can be used as three-dimensional scaffolds in the biomedical field. The disclosure describes how fibrinogen is processed under controlled conditions into fibrous structures that form a three-dimensional, biomimetic scaffold. According to the disclosure, the process enables, in particular, the controlled detachment or release of such fibrous fibrinogen scaffolds in vitro, allowing them to be detected in a solution and further used or, alternatively, immobilized on a surface. The biomaterials produced in this way are intended for medical applications, especially in the fields of wound healing and regenerative medicine, including dermal reconstruction, skin repair, vascular and bone regeneration, and tissue engineering. Applications as implant coatings are also described.A key feature revealed in the disclosure is the possibility of producing these biomaterials on demand and subsequently transferring them to a target site, such as an injury or wound area. The above problem can be solved by the present revelation, as described below. [Technical solution] In a first aspect, the present invention provides a method for producing a curved or tubular collagen scaffold, comprising the steps of: a) providing a substrate; b) cleaning the surface of the substrate; c) functionalizing the surface of the substrate by silanization using a silane or by coating it with a metal or carbon; d) depositing one or more layers of a composition comprising collagen onto the substrate; e) inducing self-assembly by adjusting the pH to form one or more layers of nanofibers comprising collagen; f) inducing coiling by fixing the one or more layers of nanofibers to form a curved or tubular nanofiber protein layer comprising collagen;g) Separating the curved or tubular nanofiber protein layer and the substrate, thereby obtaining the curved or tubular collagen scaffold, wherein step f) comprises a further step in which the fixed nanofiber protein layer comprising collagen is allowed to dry.; Surprisingly, it was found that the inventive method provides access to curved or tubular nanofiber protein layers that can be produced by spontaneously rolling up one or more layers of nanofibers comprising collagen. [Advantageous solution] The method according to the invention enables a simple and safe manufacturing process, increases reproducibility, is easy to scale, and is bio-based. Furthermore, the present invention provides spontaneously assembled rolled-up constructs made of nanofiber biopolymers, such as collagen, intended for use in tubular tissue engineering. The bio-inspired curved or tubular scaffolds according to the invention provide biochemical cues for cell adhesion and mimic the native ECM topography due to their fibrous, porous architecture. Soft tissue technology, in particular tubular tissue technology for the purposes of the present invention, can be understood as requiring the replacement of versatile tubular tissue structures after disease or injury to produce cell-informative constructs with a similar architecture that are to be implanted into the body, e.g., blood vessels, tracheal grafts, or nerve conduits. A “scaffold” for the purposes of the present invention is any support system or framework for the formation of new, viable tissue. Such a scaffold can be colonized by cells to form new tissue. A tissue scaffold produced in this way can subsequently improve or replace biological tissue, such as damaged tissue. Accordingly, the scaffold produced by the methods of the present invention can be used for medical and cosmetic purposes. A curved collagen scaffold exhibits a curvature after induced rolling of more than 5°, preferably more than 10°, more preferably more than 30°, even more preferably more than 60°, even more preferably more than 90°, and most preferably more than 180°. However, a curved collagen scaffold exhibits a curvature after induced rolling of less than 360°. A tubular collagen scaffold exhibits a curvature of at least 360° after induced coiling. In other words, the collagen scaffold is coiled into at least one full circle. The tubular collagen scaffold can be coiled to more than 360°, such as 450°, 540°, 630°, 720°, or 900°. In other words, the collagen scaffold is coiled more than once, such as 1.25 times, 1.5 times, 1.75 times, 2 times, or 2.5 times. In general, the curling of the bent or tubular framework can be determined with the naked eye. Additionally, commonly known measuring instruments can be used, e.g., light microscopy followed by image analysis for measuring the bending angle. Step a) - Providing a substrate The inventive method for producing the curved or tubular collagen scaffold is not dependent on the substrate used in step a) of the method, as long as the substrate is flexible enough to be coiled. Preferably, the substrate can be one or more materials selected from such as (porous) ceramics, polymers, or metals. Preferably, the metal is selected from gold, silver, copper, gold-plated copper, tin-plated copper, aluminum, platinum, indium, tungsten, beryllium, gallium, lithium, calcium, magnesium, zinc, titanium, zirconium, hafnium, and mixtures thereof. It is further preferred that the polymer is selected from polylactic acid (PLA), polystyrene, polybutyrate dipterephthalate (PBAT), and mixtures thereof. The (porous) ceramic is preferably selected from Al₂O₃, TiO₂, ZnO, ZrO₂, MgO, CaO, and combinations thereof. The substrate is more preferably a porous ceramic.The substrate is most preferably selected from microporous Al2O3. “Microporous” for the purposes of the present invention is defined as having a pore size in the range of 1 µm to 1000 µm. Preferably, microporous substrates or structures have a pore size in the range of 1 to 100 µm, more preferably 2 to 50 µm, even more preferably 5 to 40 µm, and most preferably 10 to 30 µm. For the purposes of the present invention, "nanoporous" is defined as having a pore size in the range of 1 nm to less than 1000 nm. Preferably, nanoporous substrates or structures have a pore size in the range of 5 to 500 nm, more preferably 10 to 300 nm, even more preferably 15 to 250 nm, and most preferably 20 to 200 nm. The pore size can be measured by scanning electron microscopy (SEM). An SEM image is used, and the pore size is obtained by a) manual analysis of a preselected number of pores or b) automated analysis. The automated analysis can preferably be performed using the ImageJ plugin BoneJ. After the manual or automated analysis, a statistical analysis is performed to obtain an average pore size. The average pore size is a numerical mean. Step b) - Cleaning the substrate The substrate is cleaned in cleaning step b) between steps a) and c) to remove grease, dirt, and / or organic residues. The substrate can be cleaned with at least one selected from the group consisting of piranha solution, sulfuric acid, nitric acid, hydrochloric acid, hexane, pentane, potassium dichromate (K₂Cr₂O₇), acetone, methanol, ethanol, isopropanol, 1-propanol, butanols such as 1-butanol, 2-butanol, and tert-butanol; ethyl acetate, diethyl ether, methyl tert-butyl ether (MTBE), methyl ethyl ketone (MEK), dichloromethane, chloroform, tetrahydrofuran, dioxane, and inert gases such as nitrogen or argon. Preferably, the substrate can be cleaned with piranha solution in cleaning step b). “Piranha solution” in the context of the present invention is a mixture of sulfuric acid and hydrogen peroxide. An example is a solution consisting of 3 parts sulfuric acid (H₂SO₄) and 1 part hydrogen peroxide (H₂O₂). The concentration of hydrogen peroxide used can be any concentration between 1% and 70%, preferably between 5% and 60%, more preferably between 10% and 50%, even more preferably between 20% and 40%, and most preferably about 30%. Such a piranha solution can effectively remove organic residues from substrates. Provided that cleaning step b) is carried out with piranha solution, it is further advantageous to subsequently dry the substrate in an inert gas stream, preferably a nitrogen (N₂) or argon (Ar) stream.Alternatively, plasma cleaning can be used in cleaning step b) to remove all organic substances from the substrate surfaces. Those skilled in the art are aware of how plasma cleaning can be carried out. For example, the substrate can be cleaned by exposing it to an ionized gas, i.e., a plasma. This process is generally carried out in a vacuum chamber using oxygen and / or argon gas. A cleaning agent can be added to one of the solutions described above. The surface of the substrate can be cleaned using an acidic and an oxidizing cleaning agent. Preferred cleaning agents are piranhaic acid and mixtures of sulfuric acid and hydrogen peroxide. Piranhaic acid is the most preferred cleaning agent. As an exemplary general procedure, substrates such as ceramic substrates were cleaned by immersion in H₂SO₅ (Piranha solution) for 5 minutes. 95% sulfuric acid (VWR International GmbH, Darmstadt, Germany) was mixed with 30% hydrogen peroxide solution (VWR International GmbH, Darmstadt, Germany) in a ratio of 3:1 to prepare fresh Piranha solution. After immersion in the Piranha solution, the ceramics were rinsed with deionized water and allowed to dry at room temperature. Step c) Functionalizing the substrate The substrate of step a) or step b) is surface-functionalized by silanization using a silane or by coating with a metal, such as gold, or carbon. In general, the metal is not restricted as long as it is suitable for coating as is generally known in the industry. Particularly suitable metals for coating are gold, silver, platinum, copper, ruthenium, rhodium, palladium, osmium, iridium, rhenium, and combinations thereof. Preferred metals for coating are gold, silver, platinum, copper, and combinations thereof. Gold is the most preferred metal. The silanes are not particularly restricted as long as they comprise at least two different functional groups, such as alkyl groups, alkoxy groups, aminoalkyl groups, aldehyde groups, mercaptoalkyl groups, biotin, ester groups, hydroxy groups, phosphonate groups, halogen groups, and mixtures thereof. The functional groups may preferably have 1 to 12 carbon atoms, more preferably 1 to 5 carbon atoms. Preferably, the silane for silanizing the substrate surface can be selected from 3-aminopropyltriethoxysilane (APTES), (3-aminopropyl)-dimethylethoxysilane (APDMES), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPS), 3-aldehydepropyltrimethoxysilane (APMS), mercaptopropyltrimethoxysilane (MPTMS), mercaptopropyltriethoxysilane (MPTES), organopolysiloxane, trimethylchlorosilane and mixtures thereof. The most preferred silane for silanizing the substrate surface can be selected from the group consisting of APTES (3-Aminopropyltriethoxysilane), DOTES (Dodecyltriethoxysilane), NPTES (N-Propyltriethoxysilane) and mixtures thereof. As an exemplary general process, a porous ceramic can be modified with (3-aminopropyl)triethoxysilane (APTES), n-propyltriethoxysilane (NPTES), or dodecyltriethoxysilane (DOTES) (Sigma-Aldrich, Darmstadt, Germany) by immersion in an ethanol solution (VWR International GmbH, Darmstadt, Germany) containing 5% APTES (Sigma-Aldrich, Darmstadt, Germany), 5% NPTES, or 5% DOTES, respectively, for 16 hours at room temperature. All modified substrates were dried and stored until further use. As an exemplary general process, coatings with a metal, such as gold or carbon, can be applied by sputter coating using the following settings: A 15 nm thick gold coating and a 10 nm carbon coating can be deposited on the textiles cleaned with piranha acid using an EM ACE600 high vacuum sputter coater (Leica Microsystems, Wetzlar, Germany). Step d) Deposition of layer(s) One or more layers of a composition comprising collagen are deposited onto the porous ceramic substrate. The number of layers is not limited. Preferably, the number of layers is 1 to 20, more preferably 2 to 15, even more preferably 3 to 10, and most preferably 3 to 5. One or more layers of a composition comprising collagen can be deposited by repeatedly applying a collagen solution to each layer. The solution containing collagen can have a concentration in the range of 0.01 mg / ml to 1000 mg / ml, preferably 0.1 to 100 mg / ml, more preferably 0.5 mg / ml to 50 mg / ml, even more preferably 1 mg / ml to 10 mg / ml and most preferably 1.5 mg / ml to 5 mg / ml. In a preferred embodiment, the layer comprising collagen may further comprise chitosan. The weight ratio of collagen to chitosan may be in the range of 20:1 to 1:1, more preferably 12:1 to 1.5:1, and most preferably 10:1 to 2:1. The one or more layers of a composition comprising collagen and further chitosan may be deposited by repeatedly applying a solution of collagen and further chitosan for each layer. The (stock) solution comprising collagen and further chitosan may have a concentration in the range of 0.01 mg / ml to 1000 mg / ml, preferably 0.1 to 100 mg / ml, more preferably 0.5 mg / ml to 50 mg / ml, even more preferably 1 mg / ml to 10 mg / ml, and most preferably 1.5 mg / ml to 5 mg / ml. The solution, which includes collagen and other chitosan, can have a concentration in the range of 0.01 mg / ml to 1000 mg / ml, preferably 0.02 to 100 mg / ml, more preferably 0.03 mg / ml to 10 mg / ml and most preferably 0.05 mg / ml to 5 mg / ml. Step e) Inducing self-organization The self-organization of the composition, which includes collagen, in step e) leads to the formation of nanofibers that include collagen. The induction of self-assembly of the composition comprising collagen is preferably achieved by the following steps: (i) adding a salt buffer and / or an aqueous buffer and / or water to the substrate or immersing the substrate in the salt buffer and / or the aqueous buffer and / or the water, (ii) drying the substrate, (iii) optionally adding a salt buffer and / or an aqueous buffer and / or water to the substrate or immersing the substrate in the salt buffer and / or the aqueous buffer and / or the water, (iv) optionally drying the substrate, (v) optionally performing one or more repetitions of steps i) to iv), thereby generating nanofibers comprising collagen. Regarding the self-organization to be induced for collagen, pH-induced self-organization may be preferable. Here, fiber formation is induced when the protein solution (which is acidic) undergoes a transition to neutral pH, for example, by adding a salt solution. When a salt buffer is added to raise the pH of the acidic collagen-based solution, the added salt buffer preferably comprises at least one component selected from sodium phosphate, sodium chloride, ammonium carbonate, ammonium phosphate, boric acid, citric acid, lactic acid, phosphoric acid, potassium chloride, potassium citrate, potassium metaphosphate, monobasic potassium phosphate, sodium acetate, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, and mixtures thereof. It is further preferred that, if the salt of the buffer is sodium phosphate, the sodium phosphate concentration is at least 1 mmol / L, preferably at least 5 mmol / L, more preferably at least 10 mmol / L, even more preferably at least 25 mmol / L, and most preferably at least 50 mmol / L.Alternatively, if the salt of the buffer is not sodium phosphate, the concentration of the required salt can be higher, such as at least 10 mm, more preferably at least 50 mm, even more preferably at least 100 mm and most preferably at least 1 M. Furthermore, it is preferred that the added salt buffer is a phosphate buffer salt solution (PBS), wherein the PBS optionally comprises sodium chloride, potassium chloride, sodium phosphate, and potassium phosphate. Preferably, the manufacturing processes of the present invention involve physiological buffers at neutral pH. Accordingly, a solution used in this process has a pH between 1 and 14, preferably between 4 and 12, more preferably between 6 and 10, even more preferably between 7 and 9, more preferably between 7 and 8.5, and most preferably between 7.4 and 8.3. The use of physiological buffer conditions has the advantage that it does not impair the biological functionality of the proteins used. The term "fiber" or "fibrous" refers to any slender, filamentous structure composed of protein components. Preferably, the term "fiber" or "fibers" refers to filamentous structures such as fibrin or collagenous connective tissue fibers. Importantly, the basic components of connective tissue are cells and extracellular protein fibers embedded in a matrix or milled substance of large carbohydrate molecules and carbohydrate-protein complexes known as mucopolysaccharides. For the purposes of the present invention, "fibrous" means any structure that consists of, contains, or resembles fibers. A structure may also be described as "fibrous" if it can be broken down into individual fibers. In a particular embodiment, the temperature preferably used in the method according to the invention can be between 15°C and 25°C, more preferably between 17°C and 23°C, even more preferably between 19°C and 21°C, and most preferably around 20°C. Furthermore, the drying of the substrate can also preferably be carried out at a temperature between 15°C and 25°C, more preferably between 17°C and 23°C, even more preferably between 19°C and 21°C, and most preferably at around 20°C. Step f) Fixing One or more layers of nanofibers are fixed to form a curved or tubular nanofiber protein layer containing collagen. Fixation is preferably achieved by cross-linking the nanofibers. In a particularly preferred embodiment, the fixing in step f) is carried out using a fixing agent selected from paraformaldehyde, formaldehyde, glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), genipin, riboflavin, mercury oxide, lead oxide, osmium oxide, trichloroacetic acid, acetic acid, and mixtures thereof. Formaldehyde and / or glutaraldehyde are particularly preferably used as the fixing agent. Glutaraldehyde is most preferably used. According to the invention, the fixing step is preferably carried out by incubating the substrate in a fixative, particularly preferably by incubating the substrate in a liquid fixative or a solution of a fixative, and most preferably by incubating the substrate in a solution of a fixative. Furthermore, it is preferred that the fixing step be carried out for at least 1 hour, preferably 12 hours, and more preferably at least 24 hours. The concentration of the fixative used can vary. More preferably, if liquid PFA is used as the fixative, the concentration of PFA is at least 1%, preferably at least 3%, and most preferably about 4%. Most preferably, glutaraldehyde is used as the liquid fixative, and the glutaraldehyde concentration is at least 0.5%, preferably at least 1%, and most preferably about 2%.Furthermore, the glutaraldehyde concentration is preferably not more than 25% and most preferably not more than 10%. After fixation, the fixed nanofiber protein layer containing collagen is preferably washed. More preferably, the fixed nanofiber protein layer containing collagen is washed with water, such as MilliQ water. The washed or unwashed fixed nanofiber protein layer comprising collagen is allowed to dry. The fixed nanofiber protein layer comprising collagen is preferably dried at a temperature in the range of 10 °C to 100 °C, more preferably 20 °C to 80 °C, most preferably room temperature. Room temperature is defined as a temperature in the range of 20 °C to 25 °C, preferably 22 °C. In a particularly preferred embodiment, the substrate can be dried overnight under a fume hood at room temperature and subsequently cross-linked for 30 minutes using 2% liquid glutaraldehyde in 1 x PBS. Aldehydrate residues can be removed in three 10-minute wash steps with deionized water. The cross-linked collagen films can then be left to dry on the substrate. Step g) Separate The nanofiber protein layer obtained in step f) can be easily separated from the substrate. Preferably, the nanofiber protein layer can detach spontaneously from the substrate if at least the nanofiber protein layer, or both the nanofiber protein layer and the substrate, are induced to curl up by the fixation process in step f). If necessary, the detachment can be assisted by a separating agent. There is no limit to the means available for separating the nanofiber protein layer. For example, tweezers can be used. An additional aspect of the present invention relates to a curved or tubular collagen scaffold obtained by the method of the present invention. A further additional aspect of the present invention also relates to a composition comprising the curved or tubular collagen scaffold produced by the method of the present invention. Another aspect of the present invention relates to the use of the curved or tubular collagen scaffold produced by the method of the present invention in tissue engineering, wound healing, regenerative medicine, nerve regeneration, skin reconstruction, skin and / or bone vessel repair, blood vessel regeneration, for fiber scaffolds, skin replacement, bone replacement, vascular prosthesis, wound dressing, implant manufacturing, implant coatings, biological filters, biosensors, as substrates for cell culture, pharmaceutical screening, pharmacological screening, toxicological screening and / or drug delivery systems. “Wound healing” in the context of the present invention is defined as a complex process in which the skin and underlying tissues repair themselves after an injury. When the skin is undamaged, the epidermis (surface layer) and dermis (deeper layer) form a protective barrier against the external environment. When this barrier is breached, a regulated sequence of biochemical events is initiated to repair the damage. This process is divided into predictable phases: blood clotting (hemostasis), inflammation, tissue growth (proliferation), and tissue remodeling (maturation). Blood clotting can be considered part of the inflammatory stage rather than a separate stage. Regenerative medicine is the support of tissue or organ regeneration. Regenerative medicine can be the process of replacing, constructing, or regenerating human cells, tissues, or organs to restore normal function. The need for regenerative medicine may arise due to an accident or injury, or because an individual, such as a patient, suffers from a degenerative disease. In a degenerative disease, cells or even entire tissues deteriorate. An example of a degenerative disease is a neurodegenerative disease, in which, for example, neurons or glial cells largely die. Cell death, in this case of neurons or glial cells, then leads to the degeneration of larger areas of affected tissue, such as certain regions of the brain. Alzheimer's disease is an example of a neurodegenerative disease. The curved or tubular collagen scaffold produced according to the invention can also be used for pharmaceutical screening. The tissue technology based on the curved or tubular collagen scaffold of the present invention enables the production of tissue in a test tube. This tissue can then be used for pharmaceutical, pharmacological, and / or toxicological in vitro studies. Based on the porous architecture, the curved or tubular collagen scaffold produced according to the invention can also be used in “biological filters” for filtration purposes in the biotechnological field. Another aspect of the present invention relates to a curved or tubular collagen scaffold produced by the method of the present invention for use as a pharmaceutical. A medical “use” in the context of the present invention preferably relates to a method for preventing or treating a disease in a subject, wherein the method comprises a step of administering a therapeutically effective amount of the curved or tubular collagen scaffold produced by the method of the present invention to the subject for use in medicine. In the context of the present invention, the term "subject" or "patient" preferably refers to a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, or monkey, or preferably a human being, such as a human patient. The subject may be suffering from or at risk of suffering from a disease. A more detailed description of medical indications relevant to the invention is provided elsewhere. Another aspect of the present invention relates to the use of the curved or tubular collagen scaffold produced by the method of the present invention for the treatment or control of a degenerative disease, a wound, a skin injury, a bone injury, a tissue disorder, a blood vessel injury, a skin disease, a vascular injury and / or a skin injury. Treatment means, for example, the treatment, delaying or alleviating of the course of a disease, the reduction of symptoms, or the cure of the disease or condition. An "effective amount" is an amount of the curved or tubular collagen scaffold produced by the method of the present invention that alleviates symptoms identified for the disease to be treated, such as a degenerative and / or skin condition. Alleviation means, for example, the prevention, treatment, and reduction of symptoms, or the cure of the disease or condition. The invention also includes a method for treating a subject at risk of developing and / or having developed a disease, wherein the subject or patient is administered a therapeutically effective amount of the curved or tubular collagen scaffold produced by the method of the present invention.A risk for the disease can result, for example, from a family history of the disease, a genotype that predisposes to the disease, or phenotypic symptoms that predispose to the disease. In one embodiment, the term "prevention" or "prevention," when used in the context of a subject, refers to stopping, hindering, and / or slowing the development or onset of a disease and, in particular, the symptoms associated with the disease. Another aspect of the present invention relates to the use of a curved or tubular collagen scaffold produced according to the invention for the manufacture of a medicament for use in the prevention and / or treatment of a degenerative disease, a wound, a skin injury, a bone injury, a tissue disorder, a blood vessel injury, a skin disease, a vascular injury and / or a skin injury. A further aspect of the present invention relates to a method for preventing bleeding at a target site in the body of a patient, wherein the method comprises delivering the curved or tubular collagen scaffold produced according to the invention to a target site, bleeding tissue, abrasion of tissue surface and / or a damaged tissue surface in an amount sufficient to inhibit bleeding. “Bleeding,” as used in the present invention, means the process of blood loss or a change in blood flow, i.e., the release of blood from the circulatory system. Bleeding can also be referred to as hemorrhage when blood is not released from the circulatory system. Another aspect of the present invention relates to a method for delivering the curved or tubular collagen scaffold produced according to the invention to a target site in the body of a patient, wherein the method comprises delivering the curved or tubular collagen scaffold produced according to the invention to the target site. In a preferred embodiment, the target site is a tissue selected from muscle, skin, epithelial tissue, connective tissue, supporting tissue, nerve tissue, tissue of ophthalmic and other sensory organs, vascular tissue, cardiac tissue, tissue of gastrointestinal organs, pleural and other lung tissue, kidney, endocrine glands, male and female reproductive organs, adipose tissue, liver, pancreas, lymph, cartilage, bone, oral tissue, mucous membrane tissue, spleen tissue, abdominal organ tissue and combinations thereof. It is further preferred that the target site be a blood site or bleeding tissue surface of a subject or patient, or a damaged tissue surface of a subject or patient. Another preferred embodiment involves the target site being a cavity within the selected tissue. It is further preferred that the cavities are selected from tissue tracts, intravertebral spaces, and / or body cavities. Another preferred embodiment relates to the target site being a tissue surface. Preferably, the tissue surface comprises an organ surface selected from the group consisting of a liver surface, a skin surface, a spleen surface, a heart surface, a kidney surface, an intestinal surface, a blood vessel surface, a vessel surface, a vascular organ surface, and combinations thereof. Another aspect of the present invention relates to a kit comprising: (a) a curved or tubular collagen scaffold produced according to the method of the present invention; (b) written instructions for applying the curved or tubular collagen scaffold to a target site on a tissue; and (c) optionally a container containing the curved or tubular collagen scaffold and the written instructions. The combination of preferred, more preferred or most preferred embodiments and / or areas that are preferred or not preferred to any extent is particularly preferred. The present invention will now be described in more detail, but not limited, by the following preferred non-limiting examples with reference to the accompanying drawings and figures. For the purposes of the present invention, all references cited herein are incorporated in their entirety. In the drawings, the figures show the following, as described below: Fig. 1 is a schematic of the method according to the invention as applied in Example 1. Fig. 2 shows the bent or tubular collagen scaffold according to Example 1. Fig. 3 shows the bent or tubular collagen scaffold according to Example 2. Fig. 4 shows the bent or tubular collagen scaffold according to Example 3. Fig. 5 shows the bent or tubular collagen scaffold according to Example 4. Fig. 6 shows the bent or tubular collagen scaffold according to Example 5. Fig. 7 shows the bent or tubular collagen scaffold according to Example 6. Fig. 8 shows an unrolled fibrinogen scaffold according to Comparative Example 4 (left, i.e., planar fibrinogen on APTES textiles) and Comparative Example 3 (right, i.e., fibrous fibrinogen on APTES textiles). Fig. 9 shows an unrolled fibrinogen scaffold according to the comparative examples 6 (left, i.e.Planar fibrinogen on bare textiles) and comparative example 5 (right, i.e., fibrous fibrinogen on bare textiles). Fig. 10 shows the curved or tubular collagen framework according to example 9. [Examples] Microscopic analysis Covered glass slides with dried frameworks were analyzed using a USB universal microscope (Meade Instruments, Rhede, Germany) at 20x magnification and brightfield imaging. For morphological analysis, dried framework samples were coated with 7 nm gold using a Bal-Tec SCD 005 sputtering system (Leica Microsystems). Scanning electron microscopy (SEM) was performed using a Zeiss Auriga field emission spectrometer (Carl Zeiss, Oberkochen, Germany) at acceleration voltages of 3 kV. Surface coverage and fiber diameter were analyzed using the open-source software ImageJ. Example 1 A microporous ceramic Al₂O₃ substrate (ALK-15 Tricot Knit Cloth; Zircar Zirconia, Inc., USA) was provided. The surface of the Al₂O₃ textile was cleaned with piranhaic acid and functionalized by silanization with APTES. Four layers of collagen were successively deposited onto the substrate by applying 100 µl of a 2.5 mg / ml collagen solution to each layer. Then, 200 µl of 10x PBS (phosphate-buffered saline) at a pH of 7.4 was applied to induce fiber self-assembly, and the assembly was left to dry overnight. Next, the nanofibers were fixed by cross-linking with 2% glutaraldehyde solution for 30 min and left to dry again overnight. During drying, spontaneous curling of the collagen layer occurred, and the scaffold exhibited a nanofiber topography (see Fig. 2). Example 2 Example 2 was carried out according to Example 1, except that the surface was functionalized by silanization with DOTES. During drying, spontaneous curling of the collagen layer occurred, and the scaffold exhibited a nanofiber topography (see Fig. 3). Example 3 Example 3 was carried out according to Example 1, except that the surface was functionalized by silanization with NPTES. During drying, spontaneous curling of the collagen layer occurred, and the scaffold exhibited a nanofiber topography (see Fig. 4). Example 4 Example 4 was carried out according to Example 1, except that the four layers of nanofibers were deposited sequentially onto the substrate by applying 100 µl of a 2.5:1 mixture of collagen and chitosan to each layer. During drying, spontaneous curling of the collagen-chitosan layer occurred (see Fig. 5). Example 5 Example 5 was carried out according to Example 1, except that the surface was coated with gold (Au). During drying, spontaneous curling of the collagen layer occurred (see Fig. 6). Example 6 Example 6 was carried out according to Example 1, except that the surface was coated with carbon (C). During drying, spontaneous curling of the collagen layer occurred (see Fig. 7). Example 7 Example 7 was carried out according to Example 1, except that the four layers of nanofibers were deposited sequentially onto the substrate by applying 100 µl of a 10:1 mixture of collagen and chitosan to each layer. During drying, spontaneous curling of the collagen-chitosan layer occurred. Example 8 Example 8 was carried out according to Example 1, except that the four layers of nanofibers were deposited sequentially onto the substrate by applying 100 µl of a 5:1 mixture of collagen and chitosan to each layer. During drying, spontaneous curling of the collagen-chitosan layer occurred. Example 9 (AAO membrane) Example 9 was carried out according to Example 1, except that a nanoporous Al2O3 membrane, i.e., anodized aluminum oxide (AAO), was used as the substrate. During drying, spontaneous curling of the collagen layer occurred, but the effect was less intense than compared to the Al2O3 textile of Example 1 (see Fig. 10). Comparison example 1 (planar collagen) Comparison Example 1 was performed according to Example 1, except that 10X PBS was not added. Thus, planar collagen was formed instead of collagen fibers. No spontaneous curling of the collagen layer occurred during drying. Comparison example 2 (no surface modification) Comparative example 2 was carried out according to example 1, except that silanization with APTES was omitted. No spontaneous curling of the collagen layer occurred during drying. Comparative example 3 (fibrinogen) Comparative Example 3 was carried out according to Example 1, except that four layers of fibrinogen nanofibers were deposited on the APTES-coated substrate instead of collagen. No spontaneous curling of the fibrinogen layer occurred during drying (see Fig. 8, right-hand sample). Comparative example 4 (planar fibrinogen) Comparative Example 4 was carried out according to Example 1, except that four layers of fibrinogen were deposited on the APTES-coated substrate instead of collagen, and no 10X PBS was added. Thus, planar fibrinogen was formed instead of fibrinogen fibers. No spontaneous curling of the fibrinogen layer occurred during drying (see Fig. 8, left sample). Comparative example 5 (fibrinogen + no surface modification) Comparative Example 5 was carried out according to Example 1, except that four layers of fibrinogen nanofibers were deposited on the substrate instead of collagen, and silanization with APTES was omitted. No spontaneous curling of the fibrinogen layer occurred during drying (see Fig. 9, right-hand sample). Comparison example 6 (planar fibrinogen + no surface modification) Comparative Example 6 was performed according to Example 1, except that four layers of fibrinogen were deposited on the substrate instead of collagen, and 10X PBS was not added. Thus, planar fibrinogen was formed instead of fibrinogen fibers. Furthermore, silanization with APTES was omitted. No spontaneous curling of the fibrinogen layer occurred during drying (see Fig. 9, left sample).

Claims

A method for producing a curved or tubular collagen scaffold, comprising the steps of: a) providing a substrate; b) cleaning the surface of the substrate; c) functionalizing the surface of the substrate by silanization using a silane or by coating with a metal or carbon; d) depositing one or more layers of a composition comprising collagen onto the substrate; e) inducing self-assembly by adjusting the pH to form one or more layers of nanofibers comprising collagen; f) inducing coiling by fixing the one or more layers of nanofibers to form a curved or tubular nanofiber protein layer comprising collagen;g) Separating the curved or tubular nanofiber protein layer and the substrate, thereby obtaining the curved tubular collagen scaffold, and wherein step f) comprises a further step in which the fixed nanofiber protein layer comprising collagen is allowed to dry.; Method according to claim 1, wherein the substrate is a porous ceramic substrate, such as microporous Al2O3. Method according to claim 1 or 2, wherein the number of layers deposited in step d) is 1 to 10. Method according to one of the preceding claims, wherein the nanofibers formed in step e) further comprise chitosan. The method according to claim 4, wherein the weight ratio of collagen to chitosan is in the range of 10:1 to 2:

1. Method according to one of the preceding claims, wherein in step e) the pH value is adjusted between 7 and 8.

5. Method according to claim 1, wherein the fixing in step f) is carried out by crosslinking using glutaraldehyde and / or formaldehyde. Curved or tubular collagen scaffold obtained according to any of the preceding claims. Use of the curved or tubular collagen scaffold according to claim 8 in tissue engineering, wound healing, regenerative medicine, nerve regeneration, skin reconstruction, skin and / or bone vessel repair, blood vessel regeneration, skin replacement, bone replacement, vascular prosthesis, wound dressing, manufacture of implants, implant coatings, biological filters, biosensors, as substrates for cell culture, pharmaceutical screening, pharmacological screening, toxicological screening and / or drug delivery systems. Use of the curved or tubular collagen scaffold according to claim 8 for preventing bleeding at a target site in the body of a patient, wherein the method comprises delivering the curved or tubular collagen scaffold to a target site, bleeding tissue, abrasion of tissue surface and / or damaged tissue surface in an amount sufficient to inhibit bleeding. Use of the curved or tubular collagen scaffold according to claim 8 as a pharmaceutical product. Use of the curved or tubular collagen scaffold according to claim 8 to target a degenerative disease, a wound, a skin injury, a bone injury, a tissue disorder, a blood vessel injury, a skin disease, a vascular injury and / or a skin injury.

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