WO2004071546A1

WO2004071546A1 - Method of producing a collagen layer

Info

Publication number: WO2004071546A1
Application number: PCT/EP2004/001374
Authority: WO
Inventors: Daniel J. MÜLLER; Jonathon Howard
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 2003-02-14
Filing date: 2004-02-13
Publication date: 2004-08-26

Abstract

The present invention relates to a method of producing a collagen layer comprising (a) introducing into a first solution located on a surface by applying a hydrodynamic flow a second solution containing solubilized collagen. It is preferred that the introduction is effected by injection or by guided directed flow. In another preferred embodiment of the invention, the method further comprises the step of (b) re-aligning the collagen fibers formed in step (a). Re-alignment may advantageously be effected mechanically by the stylus of an AFM microscope. The present invention also relates to a collagen layer producable or produced by the method of the invention and to various uses of said collagen layer including in processes of biomineralisation, cell attachment, cell motility and migration, tissue engineering, coating of implants and directed wound healing.

Description

Method of producing a collagen layer

The present invention relates to a method of producing a collagen layer comprising (a) introducing into a first solution located on a surface by applying a hydrodynamic flow a second solution containing solubilized collagen. It is preferred that the introduction is effected by injection or by guided directed flow. In another preferred embodiment of the invention, the method further comprises the step of (b) re-aligning the collagen fibers formed in step (a). Re-alignment may advantageously be effected by the stylus of an AFM microscope. The present invention also relates to a collagen layer producable or produced by the method of the invention and to various uses of said collagen layer including in processes of biomineralisation, cell attachment, cell motility and migration, tissue engineering, coating of implants and directed wound healing.

In the specification a number of documents is cited. The disclosure content of these documents including manufacturers' manuals is hereby incorporated by reference.

Collagen molecules, of which collagen type I found in bone, skin and tendons is the most common representative, are fibrous proteins composed of three peptide chains (the collagen monomers) which wind into a triple helix (the collagenmolecule) of about 300 nm in length and about 2 nm in diameter. In tissues, these molecules then self assemble into thicker fibrils whose characteristic 67-nm banding is due to the staggering of the constituent collagen molecules (Kadler, Int. J. Exp. Pathol. 74 (1993), 319-323; Kadler et al., Biochem. J. 316 (1996), 1-11 ; Eyden and Tzaphlidou, Micron 32 (2001), 287-300). Collagen constitutes up to 25% of the total protein mass in mammals and is the primary protein of the extracellular matrix. Here, if forms fibrils that resist tensile forces (Kadler, Protein Profile 1 (1994), 519-638; Hohenester and Engel, Matrix Biol. 21 (2002), 115-128). Collagen has been the subject of intense research for quite some time not only because of its role in human diseases (Kadler, Int. J. Exp. Pathol. 74 (1993), 319- 323; Prockop, Matrix Biol. 16 (1998), 519-528; Prockop, Biochem. Soc. Trans. 27 (1999), 15-31; Myllyharju and Kivirikko, Ann. Med. 33 (2001), 7-21; Kunicki, Artherioscler. Thromb. Vase. Biol. 22 (2002), 14-20) but also since it has been considered as a potentially useful biomaterial such as providing platforms for cell biological and tissue engineering applications (Guidry and Grinnell, J. Cell Sci. 79 (1985), 67-81; Bishop, Prog. Retin. Eye Res. 19 (2000), 323-344; Lee et al., Int. J. Pharm. 221 (2001), 1-22; Coombes et al., Biomaterials 23 (2002), 2113-2118). Thus, there are a number of studies making use of collagen in solution in the formation of collagen fibers. Early studies showed that explants of fibroblasts can align collagen fibers on gel surfaces. The fibers may assume a length of up to 4 cm (Stopak and Harris, Develop. Biol. 90 (1982), 383-398). Tong and Eppell, J. Biomed. Mater Res. 61 (2002), 346-353 describe the addition of collagen to a prewarmed solution of PBS. They observed a cloudy precipitate which gave rise to collagen fibers. Upon prolonged incubation, these collagen fibers became thicker, longer and often assumed an aligned order. Collagen adsorbed onto Mica was assessed by AFM. The collagen fibers thus formed were then used as a matrix for studying the formation of inorganic crystals. Hsu et al., Biomaterials 20 (1999), 1931-1936, generated microspheres comprised of collagen fibers and hydroxyapatite. The spheres were produced by discharging a collagen solution into a stirring olive oil bath with the subsequent addition of hydroxyapatite powder. It could be shown that the beads thus formed serve as an excellent support for the growth of osteoblasts and may thus find wide application in the reconstitution of bone tissue. In another set of experiments, collagen IV was used to coat electrically conducting as well as non-conducting materials. Collagen IV supported a robust growth of both glial and neuronal processes (Ignatius et al., J. Biomed. Mater. Res. 40 (1998), 264-274). Collagen has, however, the capacity to allow bacterial adhesion. This may limit the in vivo uses of the protein as a coating material for stents, for example. Tiller and colleagues (Biotechnology and Bioengineering 73 (2001), 246-252) therefore set out to chemically modify collagen. A collagen suspension was added to a Petri dish, allowed to dry and subsequently derivatised. A DMEDA-PEG derivatized collagen was found to significantly reduce bacterial adhesion. Further studies investigated the role of collagen as a coating material in the differentiation of osteoblasts (Geiβler et al., J. Biomed. Mater. Res. 51 (2000), 752-760), Becker et al., J. Biomed. Mater. Res. 59 (2002), 516-527) or corneal epithelial tissue (Evans et al., (2001) J. Biomed. Mater. Res. 56, 461-468). Collagen coating of synthetic surfaces partially overcomes growth interference of corneal epithelial tissue mediated by pores in the surfaces (Fitton et al., J. Biomed. Mater. Res. 42 (1998), 245-257.

In summary, there have been a number of approaches in the art to use collagen as a support for the growth of inorganic crystals or cells of varying origin. All these studies had the eventual goal of making use of the biomaterial properties of collagen. Pre-eminent applications of these biomaterial properties are seen, for example, in the field of osseointegration of artificial implants. Whereas the combined efforts in the past have provided significant insights regarding the characterization of collagen as a biomaterial, the state of the art suffers from the lack of options to manipulate the formation of collagen fibers in a guided fashion. This would critically expand the possibilities of using collagen as a biomaterial. The technical problem underlying the present invention therefore was to provide such options to form collagen fibers in a targeted fashion. The solution to said technical problem is achieved by providing the embodiment characterized in the claims.

Accordingly the present invention relates to a method of producing a collagen layer comprising

(a) introducing into a first solution located on a surface by applying a hydrodynamic flow a second solution containing solubilized collagen.

The term "collagen layer" denotes, in accordance with the present invention, any layer comprising collagen in an aligned fashion or collagen remodelled from an aligned fashion. This definition includes the options that the collagen is attached to a surface such as a solid surface. Alternatively, it encompasses embodiments wherein the collagen layer is not attached to a surface, for example due to removal from the surface upon carrying out the above referenced step (a). The definition further includes any type of collagen matrix such as a aligned collagen matrix or a patterned collagen matrix. Also included are collagen layers which are ultra-thin two-dimensional collagen layers or ultra-thin two-dimensionally oriented collagen matrices. Ultra-thin two-dimensionally oriented collagen layers are defined by collagen molecules, which are all oriented into the same direction and assembled into the same horizontal plane. Potentially, such well oriented layers can be used as matrix to attach secondary molecules such as proteins, nucleic acids, organic molecules, non-organic molecules or physical compounds and elements. These layers may also be used as a matrix to attach membranes, biological cells, monolayers of cells, or tissues.

The term "hydrodynamic flow" represents, as used in accordance with the present invention, a synonym for fluid dynamics and refers to any moving fluid. Fluid movement may be induced by mechanic, electric, magnetic, thermal, chemical, physical or any other effects.

The term "solubilized collagen" denotes in one embodiment collagen molecules not assembled to fibers. Also, term "solubilized collagen" comprises in accordance with the invention, all forms of collagen referred to in the art as tropocollagen. Both terms inter alia describe the collagen molecule as called the collagen triple helix. Collagen molecules spontaneously associate with each other to form collagen molecules, microfibrils and fibers. In other terms, solubilized collagen refers to the three peptide chains referred to above that are wound into a triple helix. The provision of solubilized collagen may be effected by using biological material (see references cited above or appended examples) or by means of recombinant DNA technology optionally in conjunction with appropriate purification steps. Less preferred but still possible is the production of solubilized collagen by chemical means. Thus, in principle, solubilization of collagen may be effected as described in the art referred herein or by any other suitable preparation procedures published. In addition, the term "solubilized collagen" denotes monomeric collagen, collagen molecules, collagen fibrils and microfibrils.

The term "first solution" may be an anorganic or organic solution and is preferably an anorganic solution such as a water-based solution. Preferred in accordance with the invention is that said solution displayed characteristics resembling both found in the cytoplasmic environment of a eucaryotic cell. In another preferred embodiment, the conditions present in the solution may resemble a typical extracellular environment of a eucaryotic cell; see for further guidance example 4. Also preferred is that said solution has a pH value of 7 to 8. The first solution may comprise different ingredients such as salts. Interestingly, it was found in accordance with the present invention that the nature and concentration of salts may affect the nature of the fibre/fibril formation; see for further information examples 3 to 6. Depending on the intended use of the collagen layer produced in accordance with the invention, the person skilled in the art is, on the basis of the teachings of the invention and is general knowledge in the position to adjust the pH value, the concentration of the ingredients, in particular of salts, to achieve the intended product

The term "second solution" may be the same or may be different from the first solution. The second solution may be added to the first solution or may be used to replace the first solution. It may be used to control the structural, chemical, physical or functional properties of the collagen layer. This second solution may contain different biological, organic or inorganic ingredients than the first one. It is preferred that the first and/or second solution is/are (a) buffered solution(s).

In the experimental work underlying the present invention, solubilized bovine dermal collagen was prepared and purified as described (Bell, E.₍ Ivarsson, B. & Merrill, C. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci U S A 76, 1274-8. (1979)), concentrated to » 2.5 mg/ml and stored at low pH of about 2.5. The final composition of the sample was 97% collagen type I and 3% collagen type III. Chemicals used were grade p. a. and the water was ultrapure («18 MOhm/cm). It was found, however, that any other solubilized collagen obtained from other sources, which ensured a high sample purity was suitable to achieve similar results

The controlled assembly of biological molecules is of outstanding importance for many medical and biotechnological applications. With the method of the present invention, the applications of collagen in medical and other fields can be widely expanded. It was surprisingly found in accordance with the present invention that nothing more is required for obtaining a highly ordered structure of aligned collagen fibers than the simple step of introducing into a first solution located on a surface by applying a hydrodynamic flow a second solution containing solubilized collagen. More importantly, the distance of collagen fibers as well as their thickness or the number of layers can be manipulated in accordance with the present invention. For example, the pH-value allows adjusting the density of the collagen fibers. Accordingly, increasing the pH-value results in more widely spaced collagen fibers. It was found that the electrolyte of the buffer solution has a profound effect on the collagen assembly as well. In particular, solutions containing sufficient amounts of KCI allow the collagen to establish the D- periodity of ~ 64 - 68 mm such as typically observed for thick collagen fibers. It is assumed, that collagen layers supported by other surfaces (such as stated in the specification) require different pH values to adjust their spacing and structural properties. It is also possible to vary the compositions of elecholytes to reveal a collagen layer of distinct structural and functional properties. Even more importantly and as demonstrated in accordance with the preferred embodiments further below, the collagen fibers so produced can be mechanically manipulated to create desired structures adapted to the investigator's particular needs. For simplicity the term collagen fiber used includes the formation of microfibrils, of fibrils, or of fibrillar and fibrous structures by collagen.

The avenues opened in accordance with the present invention will significantly improve the processes of collagen coating of non-biological surfaces which is required for their use in tissues implants (Steflik et al., Adv. Dent. Res. 13 (1999), 27-33; Lloyd, Med. Device Technol., 13 (2002), 18-21). One improvement, the ability to nanostructure and to align collagen arrays, may make them useful as advanced scaffolds for the attachment of additional proteins (Engel and Kammerer, Matrix Biol. 19 (2000), 283-288), and offer an avenue to direct their reaction pathways. Another improvement, the extraordinary mechanical and biochemical stability of these structures will make them useful in technologically- orientated applications, such as information storage, nanoscopic electronic circuits, or light guides which comprise stability and durability. These improvements will allow further applications including preferred applications of the collagen layer produced in accordance with the method of the invention that include coating of medical instruments and devices, implants for animal and human, protheses and artificial joints, stents and implants for the vascular system and for angioplasty devices. They further include coating of devices for treatment of cardiovascular disease, devices or elements of devices in artificial kidneys and lungs, biological materials, grafts or transplanted tissues or organs, surfaces to attach cells, cell lines and tissues. Additionally, the uses include coating of surfaces to attach proteins, inside of tubes, outside of tubes, at the end of tubes, of metals, semiconductors, non-conducting materials, plastic, wood, membranes, flexible surfaces, glass, glass surfaces, modified glass surfaces, lenses, hair, nails, skin, organs, bone, other parts of human body and cornea.

It is also a particular advantage of the present invention that the method can be implemented with a rather simple equipment as compared to prior art methods. This allows to generate the collagen layers referred to herein at a comparatively low cost.

Coating the surfaces by collagen layers will assist to change their surface properties. The collagen layers can not only be used to protect the surfaces or to enhance their biocompatibility, but also as a matrix to further coat the materials by other biological and non-biological materials. For example, it is possible to grow cells on these surfaces and to direct the cell growth by the collagen matrix, to attach proteins of desired functions, and to grow inorganic and organic material on these surfaces. Here, the collagen matrix serves as an intermediate layer which facilitates the anchoring of various materials to these surfaces and may direct the way of material assembly.

In this particular case it has been demonstrated that the properties of the collagen layer to orient cells and to direct cell movement can be tuned by the conditions at which the layers were produced and grown. For example, if the collagen layers show D-periodicity of collagen they direct and orient cells. In accordance with the present invention, it is therefore particularly preferred that the conditions are adjusted such that D-periodicity of collagen is achieved; see for further information examples 3 to 6. The collagen fibers of the ultrathin coating showed a D-periodicity between 64 - 67 nm, which is typically observed for much thicker collagen fibers (Hulmes, et al. J. Struct. Biol. 137 (2002), 2-10). In this work we found that certain buffer conditions were required to allow collagen to establish the D-periodicity. By adjusting the buffer solution (i.e. electrolyte, pH) it was possible to control whether the collagen network establishes the D-periodicty or to prevent the formation of D-periodicity. In case of lacking D-periodicity the ultrathin collagen matrices the cells attached showed different properties. It was observed, that cells of mouse fibroblasts did not show a preferential alignment. Additional these cells did not show a directed motility. In many cases the cells rounded up and detached from the collagen coating.

Additionally, the collagen layer produced in accordance with the method of the invention can be particularly used as a scaffold to functionalize non-biological surfaces, to functionalize biological surfaces, to form biological networks, to attach biological networks, to guide biological networks, to guide biological processes, to attach biological material and to attach membranes. Further, the layer produced by the method of the invention can be used as scaffold to attach lipid membranes, to attach cell membranes, to attach vesicles, to attach cells, to attach tissues, to attach DNA, to attach proteins, to attach viruses, to guide biological networks, to guide information, and to guide signals.

The collagen layer produced in accordance with the method of the invention can be further used to build artificial skin, artificial corneas, biocompatible bandages, artificial tendons, artificial cartilage, artificial lens and to structure membranes, cells, cell lines, tissues, bones, other biological materials, organic materials, non- organic materials and surfaces.

It can also be used for cosmetics, cosmetic surgery, skin grafts, skin growth, skin repair, corneal repair, growth of cell lines, to grow cells, in wound healing, in the repair of cell lines, in cancer treatment, prosthetics, artificial joints, treatment of cardiovascular disease, treatment of respiratory disorders, repair of damaged lung, repair of damaged organs, repair of replacement of tissues and for connecting non-biological with biological materials. The collagen layer produced by the method of the invention can be further used for guiding molecular processes, biological reactions, biological processes, cell movement, medical reactions, medical processes, pharmaceutical reactions, pharmaceutical processes, chemical reactions, chemical processes, biochemical reactions, biochemical processes and as templates to create nanocscopic structures, to guide electromagnetic waves, to guide photons, to guide electrons, or to guide electromagnetic information.

Additionally, the collagen layer produced by the method of the invention may be used to mechanically store information, chemically store information, biochemically store information, medically store information, store mechanic information, store chemical information, store biochemical information and to store medical information.

In a preferred embodiment of the method of the invention, said layer is a monolayer. A monolayer is a two-dimensional layer of collagen, collagen molecules, microfibrils, fibrils of fibers. In the present invention, the monolayer of aligned collagen is preferably molecularly flat, exhibits a high structural stability, and exhibits a collagen density that can be adjusted. Potentially, the monolayer can be functionalized itself or by secondary molecules and the collagen molecules of the layer can be oriented to well defined nanoscopic patterns.

In a different preferred embodiment of the method of the invention, said layer is a bilayer or multilayer. Bi- or multilayers of the collagen layers may be formed by repeating or prolonging the initial step applied to form or to manipulate a collagen monolayer. Such layers may in particular be formed in accordance with the teachings of the appended examples.

In the art, there are a number of options to apply a hydrodynamic flow in accordance with step (a) of the method of the invention. The invention relates in a further preferred embodiment to a method wherein the introduction is effected by injection or by guided directed flow.

Injection is carried out advantageously with a pipette that injects the solution containing the solubilized collagen (second buffer solution) into the physiological buffer solution (first buffer solution) covering the supporting surface. The flow direction of the injected solution (hydrodynamic flow) determines the direction of the collagen alignment on the supporting surface. With this method it is possible to align the orientation of the collagen layer in to any desired direction parallel to the supporting surface. After the collagen layer is established on the supporting surface, the sample is preferably rinsed with the physiological buffer solution (first buffer solution) containing no solubilized collagen. This last step removes loosely bound collagen molecules from the surface and stops additional collagen to adsorb from the solution onto the supporting surface.

The first solution referred to above may be any preferably aqueous solution as indicated above. It is also preferred in accordance with the method of the invention that said solution is a physiological solution.

The term "physiological solution" denotes, in accordance with the present invention, any solution that allows a biological molecule such as a protein to maintain its native conformation and optionally its natural activity. The physiological state of said solution may be determined by a variety of parameters. One important parameter is the pH-value that ranges preferably between 5 and 9, more preferred between 6 and 8 and most preferred around 7. Other parameters include ion concentrations in the solution such as salt concentration and salt type. It is preferred in one embodiment that the physiological solution has essentially the same pH value as the contents of the cytoplasma of a non diseased cell. It is preferred in a further embodiment that the physiological solution has the same pH-value and optionally the same consistency as physiologically buffered saline.

Particularly preferred is a method wherein said physiological solution is a pH- buffered solution or a redox-buffered solution.

In principle, the surface employed in the method of the present invention may be a liquid or a semi-solid surface such as a gel. However, the present invention relates in a further preferred embodiment to methods wherein the surface is a solid surface. Solid surfaces are most appropriate for the uses of the product obtained by the method of the present invention that are mentioned herein above. They include chips such as biochips of any available material, inorganic synthetic surfaces such as glass surfaces, carbon surfaces, organic synthetic surfaces such as plastic surfaces as are found in microtiter plates, biological materials such as bones, cartilage, skin, hair, teeth, lenses, cells, cell lines, tissues, cornea, metallic or non-metalic surfaces, paper, surfaces of any consistency used for implants such as calcium or hydroxyapatite based surfaces.

In a further preferred embodiment, said solubilized collagenare collagen molecules.

In another preferred embodiment of the invention, the method further comprises the step of

(b) re-aligning the collagen fibers formed in step (a).

In accordance with the present invention, it could surprisingly be shown that individual collagen molecules produced in line with the above referenced method can be manipulated mechanically, for example, by using the stylus of an AFM. The extraordinary degree to which native collagen fibers can be reorientated (Fig. 2) and reassembled into new fibers, reflect the adaptability of this extraordinary molecule to be remodelled in tissues by forces generated by fibroblasts and other cells that inhabit the extracellular matrix (Guidry and Grinnell, J. Cell Sci. 79 (1985), 67-81 ; Harris et al., Nature 290 (1981), 249-251 ; Kadler et al., Biochem. J. 316 (1996), 1-11). The option to re-align the fibers and form collagen composites of any desired structure will find wide application in the art. Hitherto it was essentially possible to provide collagen layers of some structure but the artisan had no influence of modifying the direction of the fibers after the initial formation. The AFM stylus advantageously employed in accordance with this embodiment of the invention was so far used in unrelated evaluations of biological properties such as the identification of hybridised sequence tags (Taton and Mirkin, Nat. Biotechnol. 18 (2000), 713) or bound immunoglobulin (Lee et al., Science 295 (2002), 1702-1705). Even more importantly, the mechanical manipulation of fibers may result in the generation of a molecularly continuous material which may be much longer than the initially formed fibers. The method of the present invention allows thus the formation of fibers with a length of at least 100 μm. With this method it is possible to create well-defined structures such as presently used in the microchip technology. The advantage however, is that the structures created by the method of the invention exhibit a much thinner diameter and can furthermore be functionalized by other proteins. Thus, it will be possible to use the collagen matrices as biochips to define reaction pathways of the proteins, to conduct biological, chemical and electric signals. The advantage of being able to produce long fibers up to 100 μm and more is that they can be used to establish a network that provides structural integrity, connects biological functions, or mediates cell movement and attachment. It will be also possible to use these molecular structures as templates to produce molecular conductors.

It is particularly preferred that the re-alignment is effected by applying mechanic force, hydrodynamic flow, electro-osmotic flow, electric fields, an electrochemical potential, or electrostatic interactions.

In accordance with a different particularly preferred embodiment of the invention, the method further comprises the step of stretching the fibers prior to step (b). In principle, the collagen fibers formed by step (a) may be stretched if attached, for example to a flexible support during the period of their plasticity which may last at least 2 hours, preferably at least 3 hours and more preferred at least 4 hours such as 4 to 5 hours. The elasticity of collagen molecules and fibers may be extended to much longer time scales by adjusting the environmental conditions such as the pH, electrolyte, chemical, biological, electric and physical properties of the surface supporting the collagen. Alternatively, the properties of the collagen itself may be altered to change the time scale of the elasticity. Additionally, the collagen structures may be re-plasticized after the structures have been stabilized.

For certain applications, it may be desirable to detach the collagen layer from the surface. These applications include if the supporting surface is not a solid surface but is, for example a semi-solid surface, it may be desired to remove said semi- solid surface (e.g. a gel) from the rigid collagen layer. The present invention therefore relates in another preferred embodiment to a method further comprising (c) removing the collagen layer from the surface.

Specific applications include the use of said collagen layers as adsorbing surfaces supporting the growth and/or differentiation of cells or tissues such as skin cells, hair etc.

The present invention also relates to a collagen layer producable or produced by the method of the invention.

Advantages and applications of the layer produced in accordance with the invention which may or may not be attached to a surface such as a solid surface have been outlined herein above. Some of the particularly preferred uses of the invention are outlined below.

Thus, the use of the collagen layer of the invention for directing biomineralisation is another embodiment of the invention. It is well known that the collagen matrix critically influences the process of biomineralization. Thus, our well-defined collagen matrices will allow studying the process of biomineralization in more detail and to control this process. This will be essential to control or direct the growth of artificial bones or to attach existing bones to other materials.

The invention further relates to the use of the collagen layer of the invention/producible/produced in accordance with the invention for the coating of surfaces. Coating of surfaces by collagen can be principally used to biofunctionalize these surfaces and make them suitable for biological, biotechnological and medical use. The suitability of these collagen layer coated materials to fulfil these tasks, however, depends critically on the control of the collagen layer itself. Therefore, the invention of molecular and structural well- defined collagen coatings represents a major milestone of this technique.

Preferably, the surface is an implant.

The present invention is expected to have a beneficial impact in particular in the field of implants. This is because the invention provides the options to produce ultrathin and ultraflat collagen layers, to adjust the density of the collagen within the layers, to orient the collagen within the layers over long range orders, to reassemble collagen into individual fibers exhibiting macroscopic lengths but nanoscopic widths, to molecularly pattern the collagen layer, and to adjust properties of the collagen layer by patterning and density. Importantly, the stability of the created collagen layer is extremely high which makes them suitable for many technological applications.

The use of the collagen layer producable or produced in accordance with the invention for culturing cells is also an object of the invention. An example of culturing cells on the ultrathin collagen matrices is as follows: the collagen matrices may be used for cell attachment and culturing.

Also advantageously, the collagen layer of the invention/producible/produced in accordance with the invention may be used for directing the growth of cells. An example of directing the growth of cells on the nanoscopic collagen matrices is as follows: here the aligned collagen fibrils may direct and orient the cells into tissues such as required by tissue engineering approaches.

Another object of the invention is the use of the collagen layer of the invention in the preparation of a medicinal product for wound healing.

Preferably, said wound healing is directed wound healing. An example of directed wound healing by the collagen matrices is as follows: here the aligned collagen fibrils may guide the cells involved in wound healing to grow in a certain direction.

The figures show:

Fig. 1 : Controlled self assembly of collagen onto a nearby surface. A,

Schematic representation showing the preparation method: A drop of buffer solution (pH 7.5^ 50mM Tris-HCI, 20mM NaCI, 200 mM KCI) is placed onto a freshly cleaved mica or HOPG surface. After this, the buffer solution containing a high concentration of collagen (3mg/ml) is injected into the drop. The direction of injection determined the alignment of collagen fibers onto the support. B, at pH 5.5 and lower, the collagen molecules were observed as monomers on the mica support. C, between pH 6.5 and 8.5, the collagen formed fibers which were highly aligned onto the mica surface. The alignment exhibited a long-range order up to tens of mm. D, above pH 8.5, the spacing between the collagen fibrils depends on the pH of the buffer solution. This image was recorded at pH 9.5.

Fig. 2: Controlled nanomanipulation of collagen fibrils. A, the AFM stylus was used as a tool to align collagen fibers in two rectangular areas. B, the nanomanipulated collagen fibers were aligned perpendicular to the surrounding fibrils which were assembled in two dimensions onto the supporting surface. C, after this, fibers between the rectangles could be assembled to form a "connection". All experiments were performed on native collagen molecules in buffer solution at pH 7.5. D, rectangular region of reassembled fibers showing their alignment and connection to the surrounding assembly of fibers. E, collection of fibers reassembled into different structures. Top, a single fiber extending over a length of about 4μm. Center, region of two fibers reassembled in the horizontal direction and of individual fibers forming bridges between both fibers. Bottom, two close fibers aligned horizontally. F, Different structures of reassembled fibers created by the AFM stylus.

Fig. 3: Controlled manipulation of biological scaffolds on a molecular scale.

The fibrilar structures shown represent native collagen fibers. After their parallel alignment onto a solid surface individual collagen was re-orientated with a molecular sharp stylus of an atomic force microscope to a rectangular structure (size: 500 x 800 nm). After a reaction time of four hours the collagen fibers established intra- and intermolecular covalent bonds that significantly stabilized the created biological network.

Fig. 4. Electrolyte dependent assembly of collagen. In all electrolytes used 10 mM MgCI₂ (A), 50 mM NaCI (B), 100 mM NaCI (C), 200 mM NaCI (D), 200 mM NaCI and 5 mM MgCI₂ (E), 50 mM KCI (F), 100 mM KCI (G), 200 mM KCI (H), and 200 mM KCI and 5 mM MgCI₂ (I) the collagen molecules assembled into fibrils which formed flat sheets. The thickness of the ultrathin collagen matrices was about 3 nm. Arrows indicate the direction of the fibrils within these sheets. In case of A, B and C a second layer of collagen fibrils was established onto the underlying collagen sheet, which was directly attached to the mica surface. Fibrils of this second layer had an angular orientation of -57 ± 8° relative to the fibrils under lying collagen sheet. Surprisingly, the fibrillar density of this second layer was less than that observed for the underlying collagen layer. When adsorbed in KCI (F, G, and H), the collagen fibrils were twisted around each other forming a rougher pattern. At a KCI in concentration above 200 mM (H), the collagen fibrils showed a longitudinal periodicity of 65.3 ± 2.7 nm. Adding small amounts of MgCI₂ to the monovalent electrolytes (E and I) did not influence the collagen self- assembly. All absorption buffers had a pH of 7.5 (50 mM Tris-HCI). Full gray level of topographs corresponds to a vertical scale of 5 nm.

Fig. 5. Self-assembly of collagen in buffer solutions mimicking different cellular environments. A and B, show collagen fibrils assembled on to freshly cleaved mica in phosphate buffered saline (PBS; 1.54 mM KH₂PO , 2.71 mM Na₂HPO , pH 7.4) containing 200 mM NaCI. Collagen fibrils were aligned parallel to each other covering the supporting surface almost entirely. Figures C and D show collagen fibrils assembled in buffer solution mimicking a typical cytoplasmic solution of an eukaryotic cell (130 mM monopotassium glutamate, 8.5 mM monosodium glutamate, 10 mM Hepes, 2 mM MgCI₂, 1 mM Na₂ATP, 1 mM EGTA, 0.5 mM NaH₂PO₄₍ 0.5 mM Na₂HPO₄, 0.5 mM CaCI₂, pH 7.2 adjusted with KOH). The fibrils established the characteristic longitudinal period of 66.0 ± 1.9 rim such as typically observed for collagen I fibrils of tendon and tissue. Figures E and F show collagen fibrils assembled in buffer solution mimicking a typical extracellular environment of an eukaryotic cell (109.2 mM NaCI, 4.1 mM KCI, 1.7 mM CaCI₂, 0.65 mM MgCI₂, 7.9 mM monosodium glutamate, 0.4 mM NaH₂PO₄, 0.3 mM Na₂HPO₄, 27 mM NaHCO₃, 20 mM Hepes, pH 7.4 adjusted with NaOH). Again the fibrils exposed a characteristic longitudinal period of 66.0 ± 2.3 nm. In the topographs the width of the fibrils varied from 8 nm to 284 nm (measured at FWHM), while their height above the mica was 2.7 ± 0.3 nm (π = 100). Full gray level of topographs corresponds to a vertical scale of 5 nm.

Fig. 6. High-resolution images of collagen matrices showing different properties for cell attachment. It was found that biological cells attached, aligned and were guided by collagen matrices such as shown in B. In contrast, cells did not attach well to collagen matrices shown in A. The AFM topographs were recorded in buffer solution at ambient temperatures. The thickness of the ultrathin collagen matrices was approximately 3 ± 1 nm.

Fig. 7. Mouse dermal fibroblasts align on ultrathin matrices of collagen exhibiting D-periodicity. Light microscopy images (phase contrast) showing the alignment of native fibroblasts on the aligned collagen matrices, which exhibited D-periodicity (such as shown in Fig. 6B) and coated an optically transparent glass-like surface.

Fig. 8. Mouse dermal fibroblasts do not align on ultrathin matrices lacking of collagen D-periodicity. Light microscopy images (phase contrast) showing native fibroblasts being somehow attached to the ultrathin collagen matrices lacking of D-periodicity (such as shown in Fig. 6A). The aligned collagen matrices coated an optically transparent glass-like surface. In contrast to firoblasts attached to the collagen matrices in Fig. 7 those shown in this figure were repelled from the collagen matrices after a time period of several hours.

The examples illustrate the invention.

Example 1: Controlled self assembly of collagen onto a nearby surface.

A buffer solution containing 30 μg/ml of collagen type I monomers was flushed over a mica surface (Fig. 1 A). Specifically, solubilized bovine dermal collagen was prepared and purified as described (Bell et al., Proc. Natl. Acad. Sci. USA 76 (1979), 1274-1278), concentrated to -2.5 mg/ml and stored at low pH. The final composition of the samples was 97% collagen type I and 3% collagen type III. Chemicals used were grade p.a. and the water was ultrapure (-18 MΩ/cm). 1 μl of this solution was injected into the buffer solution covering the freshly cleaved mica surface (Fig. 1A). After an adsorption time of 10 min, the sample was rinsed with a buffer solution that contained no collagen to remove loosely bound molecules. At pH-values between 2.5 and 5.5, the supporting surface was covered by individual globular particles (Fig. 1B) that presumably correspond to individual collagen peptide chains which have been previously patterned in air on solid state surfaces using the dip-pen nano-lithography technique (Lee et al., Science 295 (2002), 1702-1705). This situation changed dramatically at a pH of 6.5 and higher. In this case, the collagen molecules were observed to assemble into densely packed fibers (Fig. 1C). Surprisingly, the fibers were aligned parallel to each other over areas of more than 100 μm. The orientation of the fibers corresponded to the direction in which the collagen solution was injected into the droplet covering the support. In less densely packed regions, the average thickness of the fibers could be measured: the height of the fibers above the mica surface was 2.5 - 3.0 ± 0.5 nm (mean SD; n=214), indicating that the fibers correspond to only one or two collagen trimers. To determine the spatial extent of alignment, large mica surfaces of =20 mm in diameter were covered by the same preparation procedure. AFM topographs showed that the fibers were aligned over the entire surface. An effect of the buffer conditions on collagen self-assembly adjacent^* to a mica surface is shown in Fig. 1 D. As the pH was increased, the spacing between the collagen fibers increased and above pH 10.5 no fibrils were observed.

Example 2: Mechanical re-orientation of collagen fibers

After the native collagen fibers assembled on the supporting surface, their orientation could be manipulated mechanically. To achieve this, we used the AFM stylus as a tool to manipulate single biological molecules (Fotiadis et al., Micron 33 (2002), 385-397). The AFM (Nanoscope E, Digital Intruments, Santa Barbara, California and JPK Instruments Berlin) was operated in buffer solution using the standard fluid cell, without an O-ring. Imaging was performed in the contact mode (Mϋller et al., Biophys. J. 76 (1999), 1101-1111) or tapping mode (Mδller et al., Biophys. J. 77 (1999), 1050-1058) at an imaging force < 100 pN. The oxide sharpened Si₃N₄ cantilevers (OMCL TR400PS) employed were purchased from Olympus Ltd. (Tokyo) and had a nominal force constant of 0.09 N/m. All samples were imaged and manipulated in buffer solution at 21 °C.

First, the collagen mesh was imaged in the buffer solution in which the fibers were adsorbed. Then the imaging force of the AFM stylus was increased from 100 to 500 pN with the direction and speed of the stylus movement under operator control. After this manipulation, the area was re-imaged (Figs. 2A, B and C). As shown, the collagen fibers of the manipulated area were aligned in scanning direction of the AFM cantilever. Collagen fibers could be reoriented in any direction relative to the orientation of the initial array. Remarkably, closer observation of the native fibers (Figs. 2C-F) showed that they created a molecularly continuous material. From particular examples, such as the single fiber shown in Fig. 2E (top), it is clear, that the short collagen fiber segments were forced into a new, continuous and much longer fiber. Such observations clearly demonstrated that the AFM stylus can also direct the reassembly of collagen molecules into longer fibers. Following the described procedure, it was possible to align and to polymerise individual collagen fibers exhibiting a diameter of less than 10 nm (Fig. 2E) of up to a length of 100 μm. Close inspection of the manipulated regions shows that the collagen fibers, although exceptionally suited to withstand tensile stresses, can be bent by more than 90 degree demonstrating their excellent elastic resilience (Figs. 2D-F, Fig. 3).

The collagen arrays remained highly plastic for up to 2 hours. The structures could be continuously modified by subsequent manipulation by the AFM stylus (Fig. 2). The density, thickness and pattern of the nanocreated fibril network depend on AFM scanning parameters (i.e. applied force, scanning speed, line spacing).

The collagen arrays stabilized after 4-5 hours, after which time they could not be further manipulated. Instead, scanning with high forces resulted in the destruction of the network rather than the re-orientation and re-assembly of fibers. Concomitant with the loss of plasticity, force spectroscopy (Rief et al., Science 275 (1997), 1295-1298; Carrion-Vazquez et al., Prog. Biophys. Mol. Biol. 74 (2000), 63-91) showed that the fibers became more and more difficult to pull away from the surface. The adhesion force of single fibrils increased from -200 pN 15 minutes after formation of the oriented sheet to 1 nN after 4 hours (data not shown). Because the latter force is close to the rupture force of -2 nN measured for covalent bonds between amino acids (Grandbois et al., Science 283 (1999), 1727-1730), it is possible that the network of native collagen fibers from covalent bonds after some hours, which is within the time range found by biochemical experiments of collagen network formation (Lee et al., Science 295 (2002), 1702- 1705). Following the "setting" process, the structured collagen sheets were extraordinarily stable. When the samples were re-investigated after 1, 2 and 4 weeks of storage at room temperature in buffer solution, it was found that both the orientation and mechanical stability of the collagen fibers had not changed.

Example 3: Electrolyte dependent assembly of collagen.

To investigate the influence of electrolytes on the collagen self-assembly process, electrolyte and electrolyte concentration of the buffer solution were varied at a constant pH of 7.5 (Fig. 4). The collagen molecules assembled into fibrils under all conditions tested at neutral pH. The electrolyte, however, influenced the organization of the fibrils. The fibrils that assembled in 10 mM MgCI₂, were aligned parallel to each other (indicated by arrow) and exhibited protrusions (Fig. 4A; circles). These protrusions exhibited an average diameter of 35.7 ± 7.8 nm (n = 20) and a height of 3.5 ± 0.4 nm (n = 20) above the fibrils. Similar protrusions were also seen in 50 mM NaCI (Fig. 4B). In this case, however, the protrusions (ellipses) were elongated with an orientation (dotted arrow) of 55 ± 5° (n = 50) relative to that of the fibrils. On further increasing the NaCI concentration to 100 mM, the protrusions (ellipses) merged to form an incomplete second fibrillar layer (dotted arrow) on top of the layer of fibrils directly attached to the mica surface (Fig. 4C). The second layer made an angle of 58 ± 5° (n = 50) relative to the underlying fibrils. When the NaCI concentration was increased to 200 mM (Figs. 1 and 4E), the second layer of fibrils almost disappeared. In this condition, holes of diameter 20 - 200 nm and length 50 - 500 nm formed in the monolayer. Addition of 5 mM MgCI₂ caused theses holes to almost completely disappear, which resulted in a nearly close-packed monolayer of collagen fibrils (Fig. 4E).

Example 4: Potassium has a profound influence on the collagen assembly

Exchanging NaCI with KCI resulted in a rather dramatic change in the appearance of the fibrillar structures. At 50 and 100 mM KCI, the fibrils formed a monolayer but were not solely oriented parallel to each other (Figs. 4F and G). Although, the fibrils had an overall orientation, they appeared somehow woven and twisted around each other. Accordingly, the thickness of the resulting layers increased slightly to 3.2 ± 0.4 nm (n = 22). Further increasing the KCI concentration to 200 mM resulted in a monolayer of similar appearance (Fig. 4H). Interestingly, the modulation along the axis of fibril exhibited a periodicity of 65.3 ± 2.7 nm (n = 50). Further addition of 5 mM MgCI₂ to the buffer solution did not change the overall appearance of the fibrils forming the monolayer nor did it influence the repeat of the periodic pattern (Fig. 41).

Example 5. Assembly of collagen in buffered solutions mimicking cellular environments

In phosphate buffered saline (PBS) at pH 7.4 and containing 200 mM NaCI, the collagen molecules assembled into fibrils that covered the supporting surface forming a ultrathin matrix (Fig. 5A). The fibrils protruded 3.2 ± 0.4 nm (t? = 120) above the supporting mica surface. Higher magnification topographs (Fig. 5B), however, showed these fibrillar monolayers frequently exhibiting elongated holes. The width of the holes varied from 50 to 300 nm while their lengths extended to more than 1500 nm.

In a buffer solution mimicking the cytoplasmic environment of a eukaryotic cell (Patton H.D. et al., (1989) Textbook of physiology. W.B.Saunders Co., Philadelphia. Number of Pages 1596. Edition 21), the collagen molecules again assembled into pronounced fibrils (Fig. 5C). The fibrils protruded by about 3.8 ± 0.5 nm (π = 100) above the mica surface and exhibited a width (full-width half maximum, FWHM) varying between 5 (Fig. 5C; circle) and 650 nm. As observed before, individual fibrils laterally joined various other fibrils thereby forming a nanoscopic network. As clearly revealed from the AFM topographs, the fibrils established a pronounced longitudinal repeat of 66.0 ± 1.9 nm (average ± SD; n = 215) (Fig. 5D). The height modulation of the repeated protrusions was « 0.5 nm and did not depend on the width of fibrillar structures.

In a buffer solution mimicking a typical extracellular environment of a eukaryotic cell, the collagen molecules again assembled into pronounced fibrillar structures being aligned in the direction of the hydrodynamic flow applied (Fig. 5E). The meshlike network of fibrils was similar to that observed for the assembly in cytoplasmic buffer (Fig. 5C). In agreement to the buffer conditions mimicking the cytoplasmic environment of a eukaryotic cell (Fig. 5D), the fibrils established a pronounced longitudinal repeat of 66.0 ± 2.1 nm (n = 163) (Fig. 5F). Example 6: Cell attachment, orientation and motility can be directed by the collagen matrix

Mouse dermal fibroblasts (MDF) cell bodies aligned parallel to and elongated in orientation of the collagen microfibrils (Fig. 7). This behavior was not observed on surfaces being coated with aligned collagen lacking of the D-periodicity (Fig. 6). A finding, which was somehow surprising since the collagen layer coating the mica surface was only 3 nm thick, which corresponds to the thickness of a single collagen microfibril (Hulmes, J. Struct. Biol. 137(2002), 2-10). Although, a two-dimensional matrix can not replicate the responses of cells cultured within a three-dimensional extracellular matrix, our results clearly demonstrate that a simple substrate geometry is sufficient to control the cell orientation and attachment. MDF cells attached to surfaces coated with the ultrathin collagen matrices were observed over several hours using phase contrast video microscopy. Quantitative analysis of cell morphodynamics showed a strong correlation of cell elongation (Fig. 7) and motional directionality with the orientation of collagen microfibrils (contact guidance). In addition, MDF cells did not show directed motility on surfaces coated with collagen that lacked the characteristic 67nm D-periodicity.

Claims

1. A method of producing a collagen layer comprising a) introducing into a first solution located on a surface by applying a hydrodynamic flow a second solution containing solubilized collagen.

2. The method of claim 1 wherein said layer is a monolayer.

3. The method of claim 1 wherein said layer is a bilayer or multilayer.

4. The method of any one of claims 1 to 3 wherein the introduction is effected by injection or by guided directed flow.

5. The method of any one of claims 1 to 3 wherein said first and/or second solution is a physiological solution.

6. The method of claim 5 wherein said physiological solution is a pH-buffered solution or a redox-buffered solution.

7. The method of any one of claims 1 to 6 wherein the surface is a solid surface.

8. The method of any one of claims 1 to 7 wherein said solubilized collagen is tropocollagen.

9. The method of any one of claims 1 to 8 further comprising b) re-aligning the collagen fibers formed in step (a).

10. The method of claim 9 wherein the re-alignment is effected by applying mechanic force, hydrodynamic flow, electro-osmotic flow, electric fields, an electrochemical potential, or electrostatic interactions.

11. The method of claim 9 or 10 comprising the step of stretching the fibers prior to step (b).

12. The method of any one of claims 1 to 11 further comprising c) removing the collagen layer from the surface.

13. A collagen layer producable by the method of any one of claims 1 to 12.

14. Use of the collagen layer of claim 13 for directing biominerailsation.

15. Use of the collagen layer of claim 13 for the coating of surfaces.

16. The use of claim 15 wherein the surface is an implant.

17. Use of the collagen layer of claim 13 for culturing cells.

18. Use of the collagen layer of claim 13 for directing the growth of cells.

19. Use of the collagen layer of claim 13 in the preparation of a medicinal product for wound healing.

20. The use of claim 19 wherein said wound healing is directed wound healing.