Biocomposite and method for producing same

Abstract

The present invention relates to a method for producing oriented collagen-based biocomposite materials on the air–water interface of a solution.

Description

Biocomposites and methods for their production AREA OF INVENTION

[0001] In general, the present invention relates to biopolymer and biocomposite materials and structures, as well as to methods for their production and use. In some embodiments, the present invention is directed to oriented collagen-based biocomposite materials such as synthetic basal lamina, as well as to methods for their production. BACKGROUND OF THE INVENTION

[0002] The basal lamina (basement membrane) is a protein layer approximately 20 nm to 100 nm thick that lies adjacent to the basal pole of epithelial cells. It is a specialized extracellular matrix that connects the epithelial cells to the fibroreticular, connective tissue portion of the basement membrane. The naturally occurring basal lamina performs several functions, including influencing cell metabolism, cell spatial orientation during differentiation, and cell migration.

[0003] The basal lamina consists of two interconnected protein layers with a characteristic spatial arrangement: the lamina rara externa, an electron-permeable layer containing laminin, and the lamina densa, an electron-dense layer with collagen as its main component. The laminin forms an ordered network with a defined structure and connects the epithelial cells to the collagen.

[0004] Laminin has a cruciform structure, with the long arm of the cross consisting of a three-stranded alpha helix (alpha, beta, and gamma strands) and the three short arms (beta, gamma, and alpha chains) each consisting of a single chain. The short arms are interconnected to form a network, and the long arms establish contact with the epithelial cells. Several isoforms of laminin are known, differing in the number of alpha, beta, and gamma protein chains present.

[0005] Synthetic basal lamina, particularly its laminin coating, is used for cell cultures but can also be used for other purposes such as tissue repair materials or implant coatings like stent, occluder, or catheter coatings. Synthetic basal lamina should be biomimetic, meaning it should have the same spatial arrangement and layer thickness of laminin molecules as naturally occurring basal lamina.

[0006] Methods for producing synthetic laminin coatings on collagen layers, which can be used as a basis for synthetic basal lamina, are known. The two common methods for producing the coating are the adsorption of laminin from a solution and coating with polylaminin. In adsorption from a solution, the material to be coated is immersed in a laminin solution and removed after a certain waiting period. In the polylaminin method, acid is added, causing the laminin to cross-link in a manner similar to that found in the body. However, neither method is suitable for achieving the same orientation of the laminin as in basal lamina. Furthermore, the final layer thickness depends on a multitude of parameters, particularly the interaction between the material to be coated and the laminin, and therefore usually has to be determined empirically.Furthermore, these methods are inefficient because only a small portion of the laminin used is applied to the material to be coated.

[0007] In another method for producing such laminin coatings, cells that produce the basal lamina are cultured on the protein mixture, and then the cells are removed. This method has the advantage that the resulting coating closely resembles the natural model. However, this method is very time-consuming and expensive.

[0008] Another method for producing laminin-containing coatings for stem cell culture involves the use of Matrigel, which is derived from mouse tumors. Matrigel is a mixture of over 1800 proteins and growth factors, most of which are unknown and whose natural organization is disrupted by extraction. It adsorbs onto the surface with an unknown structure. Besides the obvious ethical concerns associated with the deliberate cultivation of tumors in laboratory animals, Matrigel has the disadvantage of being unsuitable for biomedical applications because the tissues may contain pathogens or genetic mutations and therefore pose a potential risk.

[0009] Laminin coatings can also be created by chemically attaching the molecules to the material to be coated. This requires chemically activating the material, which alters its surface properties. Reactive linkers are attached, which then react with the laminin in the next step. The unreacted linkers must then be deactivated. This process requires numerous steps and offers no control over the orientation of the laminin molecules. Therefore, a biomimetic coating is not produced using this method.

[0010] The known methods therefore have disadvantages. As described above, in some known methods the laminin does not form an ordered network because it does not orient itself according to its spatial structure; thus, no biomimetic layer is achieved. In some methods, the laminin layer thickness is not controllable and varies depending on a multitude of factors to be investigated. Parameters. Other methods are inefficient because only a small portion of the applied laminin actually binds to form a layer. A large portion of the applied laminin then has to be discarded. Further methods are time-consuming and expensive, as they require numerous process steps. Finally, some methods are unsuitable for biomedical applications because they use laminin and collagen extracted from human or animal tissue. This tissue may contain pathogens or genetic mutations and therefore poses a potential risk.

[0011] WO 2010 / 019625 A2 discloses oriented collagen-based biopolymer and biocomposite materials suitable as carriers for stem cells, as well as methods for their production. DE 11 2019 002 479 B4 discloses a method for producing a collagen-laminin matrix for the treatment of skin ulcers, burns, and wounds.

[0012] Despite considerable efforts in the development of oriented, collagen-based biocomposite materials such as synthetic basal lamina, there is still no industrial manufacturing method for these materials suitable for mass production. The present invention aims to provide a method for producing ordered collagen and laminin-based biocomposite materials as synthetic basal lamina that does not exhibit the aforementioned disadvantages. In particular, the method according to the invention should enable the simple and efficient production of oriented collagen-based biocomposite materials suitable for biomedical applications and featuring a biomimetic coating. SUMMARY OF THE INVENTION

[0013] According to one aspect, the present invention relates to a method for producing collagen- and laminin-based biocomposite materials, in particular synthetic basal lamina according to claim 1. Further embodiments are described in the dependent claims. The method according to the invention leads to novel two-dimensional biocomposite materials made of collagen and laminin, preferably of collagen IV and laminin-111, with high packing density and optimal arrangement of the laminin, in which both collagen and laminin are present in a single layer. Consequently, according to another aspect, the invention also relates to two-dimensional biocomposite materials made of collagen and laminin that can be produced according to the described method.

[0014] In the process according to the invention, an aqueous buffer solution, adjusted to a pH of 3 to 5, preferably 3.5 to 4.5, such as approximately 4, is first provided. The buffer solution can contain an alkali metal salt and / or an alkaline earth metal salt, for example an alkali metal halide such as sodium or The solution contains potassium chloride and / or an alkaline earth metal halide such as calcium chloride. An acidic collagen solution is introduced onto the subphase at the air-water interface of the buffer solution, preferably by dropwise application. The buffer solution containing the collagen solution is then allowed to stand for a sufficient time until at least some of the collagen has cross-linked to form a collagen layer at the air-water interface of the collagen solution. An aqueous laminin solution is then introduced into the subphase below the collagen layer at the air-water interface and incubated for a sufficient time until a laminin coating has formed beneath the collagen layer. Finally, the biocomposite formed at the air-water interface of the solution is transferred to a solid substrate.

[0015] Surprisingly, it was found that, using the described method, the laminin adsorbs onto the collagen layer, thus "recognizing" it as its natural environment and arranging itself (self-organizing) in such a way that the long arms of the laminin molecules point away from the collagen layer. It was also surprisingly found that the layers produced in this way resemble the natural model in their organization much more closely than layers produced by adsorption from solution or Matrigel; that the adhesion of cells to the ordered laminin molecules is stronger than in disordered laminin layers of comparable thickness; and that the requirement for collagen and laminin is minimized, since the majority of the material used is also transferred, and the thickness and organization of the coating are well-defined and independent of the material being coated.

[0016] According to one aspect of the invention, collagen IV is used as the collagen, although the invention can also be applied to any other network-forming collagens such as collagen VIII and collagen X. According to another aspect, laminin-111 is used as the laminin, although the invention is also applicable to any other laminins. Recombinant laminins and collagens can be used, which has advantages over proteins extracted from human or animal tissue.

[0017] The laminin packing density, determined by ellipsometry and calibrated for collagen IV, is preferably 250 ng crrr 2 up to 350 ng crrr 2 The value for laminin packing density can be derived from the collagen IV calibration because both are alpha-helical proteins that have similar optical properties. DETAILED DESCRIPTION OF THE INVENTION

[0018] It has been found that collagen and laminin self-crosslink in a monolayer at the air-water interface, with laminin preferentially crosslinking under acidic conditions. The higher the collagen or laminin concentration, the more this self-crosslinking is promoted.

[0019] After spreading both collagen and laminin molecules onto the aqueous solution, Brewster angle microscopy (BAM) revealed a smooth, homogeneous layer on the microscale, in which an increase in reflectivity over time was clearly visible.

[0020] The orientation and anchoring of the laminin to the collagen is also promoted by the acidic medium. According to one aspect of the invention, the collagen solution is used as an acidic solution with a pH of 3 to 5, preferably 3.5 to 4.5, such as approximately 4. According to another aspect, an acetic collagen solution is used as the acidic collagen solution.

[0021] According to a further aspect of the present invention, the collagen solution is provided with a collagen concentration of 0.1 mg / mL to 10 mg / mL, preferably 0.5 to 5 mg / mL, such as approximately 1 mg / mL, and is introduced into the subphase of the air-water interface of the buffer solution. The introduction is preferably carried out by dropwise application, for example with a pipette.

[0022] Furthermore, the Na ions can + , CI' and Ca 2+ ions present in the extracellular space of cells also alter intermolecular interactions. Monovalent ions such as Na + and CI' support the strong assembly of proteins at the interface by promoting the hydrophobic effect and shielding electrostatic interactions between the molecules. Divalent Ca 2+α-Ions are advantageous for the polymerization of laminin in bulk solution and on lipids and activate the polymerization by binding to the gamma short chains of laminin. According to a further aspect of the invention, the buffer solution further comprises alkali metal and / or alkaline earth metal salts, preferably alkali metal and / or alkaline earth metal halides. Preferably, the alkali metal salts are present at a concentration of 50 mM to 250 mM, more preferably 100 mM to 200 mM, such as about 140 mM in the collagen solution. According to another aspect of the invention, the alkaline earth metal salts are present at a concentration of 1 mM to 100 mM, preferably 10 mM to 50 mM, in the collagen or laminin solution. The alkali metal salts and / or the alkaline earth metal salts can be—each individually—either placed in the initial buffer solution or introduced into the subphase of the buffer solution together with the acidic collagen solution.The alkali metal salt is preferably selected from sodium chloride and potassium chloride, particularly sodium chloride. The alkaline earth metal salt is preferably calcium chloride.

[0023] According to another aspect of the present invention, the time required for cross-linking at least a part of the collagen to form a collagen layer at the air-water interface of the buffer solution is 30 minutes to 12 hours, for example 1 hour to 6 hours, such as about 2 hours.

[0024] According to a further aspect of the present invention, the laminin solution is preferably formulated with a laminin concentration of 0.1 mg / mL to 10 mg / mL. 0.5 to 5 mg / mL, such as 1 mg / mL, was provided and introduced into the subphase of the collagen-enriched buffer solution.

[0025] According to a further aspect of the present invention, the time required for incubating the laminin solution until a laminin coating has formed below the collagen layer is 2 hours to 24 hours, for example 6 hours to 18 hours, such as 12 hours to 14 hours.

[0026] The transfer of the formed biomimetic basal lamina to a solid substrate (support material) parallel or perpendicular to the air-water interface can be carried out using Langmuir-Schäfer transfer or Langmuir-Blodgett transfer. Both techniques are already known from the prior art.

[0027] The substrates produced according to the invention are used, for example, in science and diagnostics. Furthermore, the substrates can be used for the cultivation of stem cells. The laminin-collagen bilayers according to the invention are also suitable for the biologic modification of implants, as they promote tissue integration. The method is particularly suitable for smooth surfaces such as those found in electrodes, screws, sutures, and wires. The laminin-collagen bilayers produced according to the invention can also form defined and near-natural epithelial layers, which are suitable for pharmacological screening, for example, with regard to drug permeation and pharmacokinetics. EXAMPLE

[0028] To prepare laminin / collagen-IV bilayers, 100 pL of 1 mg / mL collagen-IV in acetic acid was first applied to 5 mL of subphase and adsorbed onto the subphase (pH = 4 with c = 100 mM NaCl) for 2 hours. Then, 50 pL of 1 mg / mL laminin was injected into the subphase using a syringe. Layer formation continued for approximately 14 hours. The collagen-IV solution was not removed or replaced before the injection of the laminin-IV solution. For transfer to a solid substrate, the layer was simply peeled off by contact with a substrate aligned parallel to the interface, using the Langmuir-Schäfer transfer method. Fluorescence microscopy confirmed that the layers were separate, as the fluorescence of laminin and collagen did not colocalize.

[0029] To demonstrate that the amount of protein deposited is constant with each transfer, stacks of 1, 3, and 5 layers were fabricated on Si wafers and gold-coated glass supports and characterized by PM-IRRAS, ellipsometry, and AFM. The thickness of a Lam-111 2D network, determined by AFM, was (3.2:1:1) nm. The network-like morphology of the Lam-111 2D network on the Si wafer is clearly visible in the AFM phase image, while the height image shows a uniform thin layer (all features below 5 nm height). For 5 layers, the thickness was four times that of a single layer at (14.8:1:2.1) nm. The thickness was This was confirmed by ellipsometric measurements, in which the thickness of the multilayers was determined to be (8.9:1:0.1) nm for 3 layers and (16.1:1:0.3) nm for 5 layers. The thickness of the Laminin-111 monolayers could not be directly determined ellipsometrically without knowledge of the layer's refractive index, as the thickness is below the ultrathin film limit. The packing density of the fabricated basal lamina, determined by ellipsometry, was 315 ng crrr after approximately 2 hours. 2 or 3.7-10 13 mol crrr 2 Precise control of the areal protein concentration was achieved by PM-IRRAS of laminin-111 films on gold-coated glass, performed after each transfer in a multilayer deposition experiment. A stepwise linear increase in protein concentration (integrated amide I and amide II intensity) was observed with each deposition step.

Claims

Patent claims 1. A process for producing biocomposite materials from collagen and laminin comprising the steps: Providing an aqueous buffer solution adjusted to a pH of 3 to 5; Introducing an acidic collagen solution into the subphase at the air-water interface of the buffer solution Allowing the buffer solution containing acidic collagen solution to rest for a sufficient time until at least part of the collagen has crosslinked to form a collagen layer at an air-water interface of the collagen solution Introducing an aqueous laminin solution into the subphase below the collagen layer at the air-water interface and allowing the laminin-added buffer solution to rest for a sufficient time until a laminin coating has formed below the collagen layer Transferring the biocomposite formed at the air-water interface of the solution to a solid substrate.

2. The method according to claim 1, wherein the acidic collagen solution used is a collagen IV, collagen VIII, collagen X solution or a mixture thereof.

3. A method according to claim 1 or claim 2, wherein the laminin solution used is a laminin III solution.

4. A method according to any one of the preceding claims, wherein the acidic collagen solution is introduced into the subphase at the air-water interface of the buffer solution by dropwise addition.

5. A method according to any one of the preceding claims, wherein the buffer solution mixed with acidic collagen solution is allowed to rest for a period of 30 minutes to 12 hours, preferably 1 hour to 6 hours, particularly preferably about 2 hours.

6. A method according to any one of the preceding claims, wherein the aqueous laminin solution is introduced into the subphase beneath the collagen layer at the air-water interface by means of a syringe.

7. A method according to any one of the preceding claims, wherein the buffer solution containing laminin is allowed to stand for a period of 2 hours to 24 hours, preferably 6 hours to 18 hours, particularly preferably 12 hours to 14 hours.

8. A method according to any one of the preceding claims, wherein recombinant collagen and recombinant laminin are used.

9. The method according to any one of the preceding claims, wherein the buffer solution further comprises alkali metal and / or alkaline earth metal salts, preferably alkali metal and / or alkaline earth metal halides.

10. The process according to claim 9, wherein sodium chloride and / or potassium chloride is used as the alkali metal halide.

11. A process according to claim 9 or 10, wherein calcium chloride is used as the alkaline earth metal halide.

12. Method according to one of the preceding claims, wherein the collagen solution is provided with a collagen concentration of 0.1 mg / mL to 10 mg / mL, preferably 0.5 to 5 mg / mL.

13. Method according to one of the preceding claims, wherein the laminin solution is provided with a laminin concentration of 0.1 mg / mL to 10 mg / mL, preferably 0.5 to 5 mg / mL.

14. Biocomposite material made of collagen and laminin, producible by a process according to any one of the preceding claims.

15. Biocomposite material according to claim 14 having a protein packing density of 250 ng crrr 2 up to 350 ng crrr 2 , preferably 300 ng crrr 2 up to 325 ng crrr 2 determined by ellipsometry, calibrated for collagen IV.