Field

The disclosed inventions relate to a method of cross-linking biopolymer materials, like proteins, with a polyfunctional aziridine cross-linking agent, to methods of making a coating or an article comprising a cross-linked biopolymer like a collagen, and to products comprising a cross-linked biopolymer obtained with said methods, like a collagen-based coating on a medical device.

Background

Biomaterials based on natural biopolymers like proteins (e.g. collagens, silk) and polysaccharides (e.g. cellulose, starch) or based on synthetic biopolymers like (co)polyesters derived from lactic acid are generally preferred for use in biomedical applications, e.g. as scaffolds for tissue engineering or as particles for controlled drug release, but often lack the mechanical properties and stability required for use in aqueous environments. One approach toward improving properties of such biomaterials for use in in vivo conditions is to stabilize the material by (chemically) cross-linking the biopolymer.

An article by Reddy et al. (DOI: 10.1016/j.tibtech.2015.03.008) provides a review of methods applied for making cross-linked biomaterials from natural and synthetic biopolymers. As cross-linking aims to enhance mechanical properties and stability in aqueous environments needed for use in medical applications, the article discusses cross-linking approaches starting from the physical form of the biomaterial; i.e. as films, hydrogels, porous scaffolds,

(electrospun) fibers, and nanoparticles. Commonly applied cross-linking methods apply compounds like glutaraldehyde (GA), genipin, polycarboxylic acids, or a combination of ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) as cross-linking agent. It is concluded that properties of biomaterials can be significantly improved, but that handleability, side reactions and/or cytotoxicity of the cross-linking agents used generally pose problems; and thus, that new cross-linking technologies need to be developed for making biopolymeric materials that are suited for medical applications.

Krishnakumar et al. discuss in a review article (DOI: 10.1016/j.msec.2018.11.081) different cross-linking mechanisms as applied in methods of making 3D scaffolds from a protein biomaterial like collagen; aiming to improve properties for use of the scaffold as a template for bone tissue engineering. Again, it is indicated that the most frequently applied methods use GA, genipin or EDC/NHS as cross-linking agent. Other cross-linking strategies apply a.o. 1 ,4- butanediol diglycidylether (BDDGE) as cross-linker or use enzymatic approaches. The authors conclude that an ideal cross-linker resulting in a material with optimal properties for bone-like tissue synthesis, providing the desired biocompatibility and no cytotoxicity is still 'impractical'.

US4772285 describes a soft tissue prosthesis like a breast implant made from silicone polymer, which is provided with a collagen coating to prevent encapsulation of the implant by fibrous tissue. An aqueous collagen coating composition is made, for example from Type I collagen from which telopeptides have been enzymatically removed. This collagen coating composition is applied to the surface of the implant, subsequently dried, washed with e.g. acetone and water, and then cross-linked by placing in an aqueous bath of formaldehyde or glutaraldehyde.

In US4842575 a collagen-impregnated synthetic vascular graft, for example a warp knit polyester fabric, is made by depositing an aqueous slurry of collagen fibers on the graft and mixing the collagen into the porous structure of the graft, followed by drying and cross-linking to result in a substantially non-porous, blood-tight graft. The collagen is thereafter cross-linked by exposing to formaldehyde vapor followed by vacuum drying.

US2001/0034550A1 describes a metal or other stent for vascular implantation, which stent has an outer collagen coating and/or an inner collagen liner. Such coating and/or liner provides an endovascular stent which protects the vascular wall and forms a non-thrombogenic cushion for the stent. Collagen may be in the form of a sleeve per se, be carried on a support like a polyester fabric, or be applied as a coating directly on the stent. The term collagen is defined in this publication to not only relate to polypeptide structures known as collagens, but to also include other natural materials normally forming membranes with collagen like laminin, keratin, elastin, glycoaminoglycans, carbohydrates, fibronectin, hyaluronic acid and the like.

The collagen sleeve or coating can be made from aqueous collagen compositions that may further contain pharmaceutical agents like heparin or anti-oxidants like vitamin E. Elasticity of the collagen sleeve may be adjusted by altering the cross-link density thereof; for example by radiation exposure, heating, or by chemical cross-linking agents like dialdehydes, formaldehyde, N-hydroxy-succinimide esters or diacid chlorides.

US9186432B2 relates to a high-strength surgical suture comprising UHMWPE fibers and provided with a collagen coating, which suture may be used in surgical procedures like tendon repair wherein the collagen coating aids in the tendon-to-bone incorporation process by stimulating proliferation and protein synthesis. The coating may be provided by soaking a suture in a diluted acidic solution of collagen, followed by drying in air. There is no mentioning of cross-linking the collagen.

In US2010/0119605A1 collagen cross-linking/stabilization compositions are described, optionally in combination with an elastin cross-linking composition, for use in in vivo treatment of vascular aneurysms. The treatment is achieved through the delivery of an effective amount of the cross-linking/stabilization composition to the site of the aneurysm. Various cross-linking agents are mentioned as being suitable for such purpose, including glutaraldehyde and other bifunctional aldehydes, diamines/carbodiimides, genipin, and epoxy compounds like triglycidyl amine.

US2012/0220691A1 discloses a photo-cross-linkable material based on type-l collagen, the mechanical properties of which may be modulated by light-induced cross-linking. Collagen methacrylamide is made by reacting free amines of collagen with methacrylic acid and a carboxylic acid activating reagent like NHS, in the presence of a carbodiimide such as EDC. The process would not significantly alter the structural and functional properties that make type I collagen an attractive and useful scaffold material. The obtained methacrylated collagen retains the ability to self-assemble from a liquid macromer solution into a fibrillar hydrogel at physiological pH and temperature, with similar assembly kinetics and resultant structure as compared to native type I collagen.

US2013/0226314A1 relates to making biocompatible tissue-based membranes that are minimally adhesive on both sides when used for tissue repair in medical and dental surgery. The method for making smooth tissue-based coated membranes comprises steps of coating a processed and smoothened tissue-derived fibrous membrane, for example based on bovine pericardium, with collagen, and exposing the coated membrane to a cross-linking agent. Coating may be done by conventional means with an aqueous dispersion of collagen fibers, followed by drying and cross-linking the coating layer with for example a solution or vapor of glutaraldehyde or formaldehyde.

Meng et al. (DOI: 10.1039/c2jm31618h) describe in-situ cross-linking of collagen compositions during electrospinning. The compositions used comprise lyophilized acid-soluble collagen Type I, EDC (carbodiimide) and NHS (N-hydroxy succinimide) at molar ratio NHS/EDC >1.5, and ethanol/PBS (50/50) as solvent. In-situ cross-linking in this case means that the gelation time of the solution, typically from several to 50-200 minutes allows electrospinning into fibers and subsequent cross-linking of the fibers formed without addition of further components or other post-production cross-linking process to generate water-insoluble collagen nanofibers.

In US2004/0143180A1 a medical device with a hydrogel coating is described, wherein the coating includes a MRI contrast agent and has inner and outer regions comprising a first and second hydrogel polymer, respectively. Examples of hydrogel polymers include natural polysaccharide and polypeptide biopolymers, like alginic acid and collagens. The outer region may comprise an ionic cross-linking agent and the inner region a covalent cross-linking agent, which can be a polyfunctional aziridine compound. In experiments, inner hydrogel coatings were made from compositions comprising sodium alginate and a commercial polyfunctional aziridine compound (Crosslinker CX-100; DSM Resins). Summary

Although various approaches have been proposed or described in literature, there still appears a need for a method of cross-linking natural and synthetic biomaterials, for example of collagens, which method can be applied under mild conditions not deteriorating the biopolymer, does not pose problems regarding biocompatibility and toxicity, and which method results in improved properties and performance of cross-linked biopolymer.

Objects of the present disclosure include providing a method of making cross-linked biopolymers, which addresses one or more of said needs .

The aspects and embodiments as described herein below and as characterized in the claims provide a method of cross-linking biomaterials containing natural or synthetic biopolymers, by reacting functional groups of the biopolymer with a polyfunctional aziridine cross-linking agent AZ, which compound a) comprises from 2 to 6 of the following structural units (A): wherein Ri is H;

R and R ₄ are independently chosen from H, a linear group containing from 1 to 8 carbon atoms and optionally containing one or more heteroatoms in the chain;

R ₃ is a linear group containing from 1 to 4 carbon atoms and optionally containing one or more heteroatoms in the chain;

R’= H or an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms;

R” = H, an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms, a cycloaliphatic hydrocarbon group containing from 5 to 12 carbon atoms, an aromatic hydrocarbon group containing from 6 to 12 carbon atoms, CH ₂ -0-(C=0)-R’”, CH ₂ -0-R””, or CH ₂ -(OCR””’HCR””’H)n-OR”””, whereby R’” is an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms and R”” is an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms or an aromatic hydrocarbon group containing from 6 to 12 carbon atoms, n being from 1 to 35, R’”” independently being H or an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms and R””” being an aliphatic hydrocarbon group containing from 1 to 4 carbon atoms; whereby R’ and R” may be part of the same saturated cycloaliphatic hydrocarbon group containing from 5 to 8 carbon atoms; and m is an integer from 1 to 6; b) comprises one or more linking chains wherein each one of these linking chains links two of the structural units (A) present in the polyfunctional aziridine compound; and c) has a molar mass in the range of from 600 Da to 5000 Da.

This method can advantageously be performed at conditions compatible with natural biopolymers, for example without inducing denaturation of proteins like collagens, the method does not require addition of further reactive components, results in cross-linked materials having improved physical properties, good stability under physiological conditions, and suitable biocompatibility. A specific advantage of the method is that the new cross-linking agents AZ have been reported to be non-genotoxic or at least to show markedly reduced genotoxicity compared to commonly applied aziridine compounds comprising trimethylolpropane tris(2-methyl-1-aziridineproprionate; see W02020/020714A1 (published after the first filing date of present application). The present method introduces cross-links in the biopolymer by reaction of aziridine end groups of the cross-linking agent with functional groups, like pendant nucleophilic groups, of the biopolymer. This reaction can for example be performed in aqueous environment at temperatures between 0 and 80 °C. Especially in case the biopolymer has pendant carboxyl groups with active hydrogens, the reaction of biopolymer with the aziridine compound may be performed at ambient conditions, like at 0-35 °C. Such conditions may be below the denaturation temperature of many proteins; and can thus for example prevent -often irreversible- disruption of the 3-dimensional structure of a protein, such as the complex structure of fibrous collagen containing aggregates of tri-helical fibrils, during said cross-linking reaction.

In another embodiment, the disclosure provides a method of making a coating comprising a cross-linked biopolymer on a substrate, comprising steps of

• Making a solution or dispersion of biopolymer material;

• Mixing a polyfunctional aziridine compound AZ into the solution or dispersion;

• Applying the obtained mixture to a surface of the substrate to form a coating;

• Drying the formed coating, and

• Cross-linking the biopolymer in the coating before, during and/or after drying by reacting functional groups of the biopolymer with aziridinyl groups of the polyfunctional aziridine compound AZ.

Analogous to the general cross-linking method, such coating method can be performed at mild conditions compatible with natural biopolymers, for example in aqueous medium and/or without inducing denaturation of proteins like collagens, does not require addition of further components, does not apply compounds that result in toxicity risks of the modified biopolymer, and produces cross-linked material having improved properties and stability under physiological conditions. The obtained coating furthermore shows good mechanical properties and good adhesion to the surface of the substrate, such as a metal surface. The coating may be suitably applied to various substrates, especially to medical devices, which may be made from different materials, including metals and organic polymers, and may have different physical shapes; including non-porous, monolithic implants like titanium screws and porous structures like fibrous constructs such as sutures or fabrics comprising polyester or polyethylene yarns.

The cross-linking method and/or the coating method may also be applied as a sequence of subsequent steps to form an article, like in an additive manufacturing or 3D-printing process.

In a further aspect, the disclosure provides an article or a component comprising a cross- linked biopolymer as can be obtained with the above methods, like a porous biomaterial comprising a collagen-based sheet or other structure for use in for example tissue engineering.

In another aspect, this disclosure provides a medical device or medical implant, comprising an article or a component comprising a cross-linked biopolymer as can be obtained with the above methods, like an orthopedic implant having a collagen-based coating on at least part of its surface.

Brief description of Figures

The invention will be further elucidated by the following illustrative figure, without being restricted thereto.

Figure 1 represents a photo of 5 test plates, each plate having coated areas made from the collagen compositions of Ce1 , Ex 2, Ex 3 and Ce 4 (from left to right on each plate), after being submitted to tests 1-5.

Detailed Description

Within the context of present disclosure, a biocompatible material is biologically compatible by not producing a toxic, injurious, or immunologic response when in contact with living tissue. Biodegradable means that a material is susceptible to chemical degradation or decomposition into simpler components under physiological conditions or by biological means, such as by an enzymatic action. Biostable or bioinert means that the material is substantially non-biodegradable under conditions and time of intended use in contact with living tissue.

A biopolymer material, also called biomaterial, is understood to comprise (or to be substantially composed of) a biopolymer that can be i) a natural polymer that has been biosynthesized by a living organism or which has been derived from a natural source, or ii) a synthetic polymer that has been made at least partly from monomers occurring in nature and/or produced from renewable feedstock; and which polymers are biocompatible and may be biodegradable or biostable.

Collagen is the major insoluble fibrous protein in the extracellular matrix (ECM) and in connective tissue. There are at least 16 types of collagen, but 80-90 percent of the collagen in the body consists of types I, II, and III. The collagen polymer molecules can pack together to form triple helices that arrange into long thin fibrils. Collagen types may associate with each other and/or with other ECM components. Within present disclosure, collagen is understood to refer to all such types of biopolymers, and which biopolymer material may comprise further compounds like other ECM components.

A polyfunctional cross-linking agent is a compound having multiple, that is at least two, reactive groups that can react with functional groups of a biopolymer to form (cross-)links within and/or between oligomeric or polymeric chains of the biopolymer material. For example, a polyfunctional aziridine cross-linking agent is a compound having two or more aziridine groups; such compound is also referred to as a multi-aziridine compound. An aziridine group (also called aziridinyl group) is a three-membered heterocycle with one amine (-NR-) and two methylene bridges (-CR -), which group can react in ring-opening reactions with nucleophiles, for example with a carboxyl or a thiol group, due to its ring strain.

A compound that is cytotoxic is toxic to living cells, genotoxic means a compound can cause damage to DNA. Within present application, a compound is called non-genotoxic if it shows a negative induction level, that is less than 1.5-fold induction, for the biomarkers at all concentrations as determined with the ToxTracker® assay (see e.g. W02020/020714A1).

In accordance with an aspect, the invention provides a method of cross-linking a biopolymer material; the method comprising a step of reacting functional groups of the biopolymer contained in the biopolymer material with aziridine groups of polyfunctional aziridine compounds AZ (as defined herein) as cross-linking agent.

Chemically cross-linking a polymer generally results in forming a very large 3-dimensional polymeric molecule or polymer network, depending on the form of the biopolymer during the cross-linking reaction. As further processing into another desired physical form or shape of a cross-linked polymer is generally difficult or even practically impossible, the biopolymer, for example as a biopolymer composition like a solution of dispersion of the biopolymer (material) in aqueous medium, is preferably already in the physical form or shape that is desired for the targeted application when it is reacted with the aziridine cross-linking agent. Examples of physical forms include 3-dimensional, optionally porous structures that may be applied as a template or scaffold for tissue engineering; nano- or microparticles that may for example by used for controlled release or delivery of a bioactive agent contained in such particles; and thin (coating) layers on a surface of a biomedical device like an orthopedic implant, a stent or a fibrous suture.

In embodiments, the invention thus also provides a method of making an article comprising a cross-linked biopolymer, the method comprising steps of forming a biopolymer composition into an article of a desired shape, adding a polyfunctional aziridine compound AZ (as defined herein) to the biopolymer (composition) before, during and/or after the forming step, and reacting the biopolymer with the polyfunctional aziridine compound to make an article comprising the cross-linked biopolymer, optionally in the form of a hydrogel; e.g. when starting from a biopolymer composition like a solution of dispersion of a hydrophilic biopolymer in aqueous medium.

In further embodiments, the disclosure for example provides a method of making a coating comprising a cross-linked biopolymer on a substrate, the method comprising steps of

• Making a solution or dispersion of a biopolymer material;

• Mixing a polyfunctional aziridine compound AZ (as defined herein) into the solution or dispersion;

• Applying the obtained mixture to a surface of the substrate to form a coating;

• Drying the formed coating, and

• Cross-linking the biopolymer in the coating before, during and/or after drying.

In embodiments, the cross-linking method and/or the coating method may be repeated multiple times. By repeating the coating method, for example, a multi-layer coating may be formed layer-by-layer, or a 3-dimensional article wherein a formed layer functions as substrate for a subsequent layer can be made. Such layer-by-layer methods are also referred to as additive manufacturing or 3D-printing processes.

It is further noted, that in addition to reaction of functional groups of the biopolymer with the polyfunctional aziridine compound to form cross-links, also reaction between aziridine groups and a functional group present on a surface of a substrate may take place; to result in (cross-linked) biopolymer being chemically bound, also called grafted, to the surface. In general, it is an advantage of present method that a coating made therewith may show good adhesion to the surface of various substrates, like a metal surface.

The biopolymer material used in the methods of present disclosure can be based on a natural polymer or a synthetic polymer. Within the context of this application a biopolymer may have a molar mass than can vary widely, and may be oligomeric with relatively low molar mass, like oligo peptides or protein fragments, or a high molar mass polymer. Within present application an oligomeric biopolymer may have a molar mass of about 200-10.000 g/mol or 300-5.000 g/mol; and a polymer typically has a molar mass of at least 5.000, 10.000, 15.000 or 20.000 g/mol. Molar mass of a biopolymer may be limited for practical reasons, and is typically at most about 2.000.000 g/mol or at most 1.000.000 or 500.000 g/mol.

In embodiments, the biopolymer material comprises or substantially is a natural biopolymer. Generally, three main classes of natural biopolymers are distinguished, i.e. proteins, polynucleotides, and polysaccharides, but also bacterial polyesters, natural rubber and lignin are natural polymers.

Proteins are polypeptides, i.e. copolymers comprising different amino acids as their monomer units that are linked together via peptide bonds. Amino acids are distinguished by their side groups, which comprise amines, carboxylates, hydroxyls, phenolics and sulfhydryls. Type, number, and sequence of the various amino acids determine molar mass, hydrophobicity, electrical charge, physical interactions, chemical reactivity, and structural conformation of a polypeptide. Proteins are generally of high molar mass and may have very specific 3-dimensional conformations, like a globular structure wherein a polypeptide chain is folded into a compact spheroid conformation, or a fibrous structure based on aggregated fibrils formed from three helically-ordered peptide chains characteristic for collagens. The biopolymer can be a complete protein, for example TGF-b, FDF, CTGF, PGE-2, TNF-a, PDGF, IFNg, BMP-2, interleukins, but also a smaller (that is, a relatively low mass) peptide or protein fragment like RGD or P15.

Polynucleotides are polymers having different nucleic acids as monomer units, like DNA and RNA. Intermolecular interactions between nucleotide result in ordered structures, like the DNA double helix.

Polysaccharides are oligomeric or polymeric carbohydrate structures based on different monosaccharides or sugars as monomer units. Like proteins, polysaccharides may have different pendant functional groups and specific structural conformations like helical structures, dependent on their composition. Examples of polysaccharides include cellulose, the most common organic compound and biopolymer, starch, heparin, alginates, carrageenans, chitin, chitosan, and their derivatives.

In further embodiments, the biopolymer material comprises or substantially is a synthetic biopolymer, such as (co)polymers of lactic acid and glycolic acid like PLA and PGLA, and polyhydroxybutyrates (PHB). Unlike in most natural biopolymers, different monomers in synthetic copolymers are generally randomly distributed along the polymer chain. Such polymers may have different functional groups, as a group pendant on the chain and/or as an end group of the polymer chain.

In embodiments of present invention, the biomaterial or biopolymer used in the method comprises or is a protein. In further embodiments, the biomaterial comprises, or is, a collagen; like a fibrous collagen, an acid-soluble collagen, or mixtures of different collagens. In other embodiments, the biomaterial or biopolymer used in the method comprises fibrous collagen. In further embodiments, the biopolymer used in the method is a fibrous collagen. In further embodiments, the biomaterial or biopolymer comprises one or more collagen types and elastin.

In embodiments, the biopolymer comprises elastin or a soluble collagen, for example a bovine-derived collagen powder like Semed S, which comprises about 95% type I and 5 % type III collagen (obtainable from DSM Biomedical Inc., Exton (PA), USA). In US2016/0129151A1 a method is described, wherein such collagen is dissolved in a benign solvent, formed from water, alcohol and salt; and from which solution the collagen can be precipitated by adding more alcohol.

In the methods of present invention, the biopolymer comprises one or more nucleophilic groups as functional groups, such that cross-links are made in the biopolymer by reaction of aziridine groups of the cross-linking agent with nucleophilic groups of biopolymer chains, which nucleophilic groups may be pendant groups or end groups. Such nucleophilic ring-opening of the heterocyclic aziridine ring is known in the art, see for example the review article by Hu (DOI: 10.106/j.tet.2004.01.042).

In embodiments, the biopolymer comprises at least one hydroxyl, amine, thiol, sulphonic acid, or carboxyl group, like at least 2, 4, 6, or 8 nucleophilic groups. In embodiments, the biopolymer comprises at least one carboxyl group, preferably multiple carboxyl groups, like at least 2, 4, 6, or 8 groups. Considering a biopolymer may have a specific conformation, at least some of the nucleophilic groups may be ‘hidden’ within the biopolymer structure and not available for reaction. In general, not all nucleophilic groups present need to react with the cross-linking agent for effectively cross-linking the biopolymer. One of the advantages of present methods of cross-linking a biopolymer is that the reaction with the aziridine-functional compound can be performed in an aqueous environment and at ambient or higher temperatures, for example between 0 and 80 °C or between 0 and 35 °C. Such relatively mild conditions allow controlling the specific 3-dimensional conformation that a biopolymer may have, and which conformation may preferably be maintained upon cross-linking, depending on targeted use. Such reaction conditions for example enable preventing (complete) denaturation of a protein like collagen; and to retain at least the triple helix structure of collagen fibrils. Especially the reaction of the aziridine compound with active hydrogens of carboxyl groups of the biopolymer may be suitably performed at ambient conditions, like at 0-35 °C, which would be below the denaturation temperature of many proteins; and can thus for example prevent disruption of the triple helix structure of collagen fibrils or even of collagen fibers comprising such fibrils. Within the context of this disclosure, a sulphonic acid group or a carboxyl group is understood to include such an acid group and derivatives thereof that can react or be made to react with aziridine, like esters, anhydrides or salts.

In embodiments, the biopolymer material may contain in addition to polymer chains further components, generally organic components, for example relating to the process with which the biopolymer material, especially a natural biopolymer, was obtained. If the material was derived from a natural source, such as a collagen composition extracted from porcine or bovine hide or skin, it may in addition to biopolymer for example contain other ECM components originally present in the source and/or residual components used in the extraction or purification processes. In other embodiments a further component may have been added to the biopolymer, such as a processing aid, or a bioactive agent like an antimicrobial or antibacterial agent. Such components may also be added to a solution or dispersion of biomaterial or a biopolymer composition.

In other embodiments, the biopolymer composition or the solution or dispersion of biopolymer material comprises inorganic particles like bioceramic particles such as a calcium phosphate like hydroxyapatite as further components; which particles are typically dispersed in such composition.

The amount of further components in the biopolymer material may vary considerably, depending on the type of further component; like from 0.1-90 mass% (based on total mass of biopolymer and further components; or based on total mass of biopolymer material). In embodiments, the further components are organic components that may be present from 0.1 to 10 mass%; preferably the amount is at least 0.2, 0.4, 0.6, 0.8, 1 or 2 mass% ) and at most 9, 8, 7, 6, 5, 4, or 3 mass%.

In other embodiments, the further components are inorganic components like bioceramic particles, such as bioglass and/or calcium phosphates like hydroxyapatite, which particles may be present from about 5 to about 90 mass% (based on total mass of biopolymer and further components); preferably the amount is at least 10, 15, 20, or 25 mass% and at most 85, 80, 75, 70, 65, 60, 55 or 50 mass%.

In embodiments, the biopolymer material further contains a compound selected from cell signaling moieties, moieties capable of improving cell adhesion, moieties capable of controlling cell growth (such as stimulation or suppression of proliferation), antithrombotic moieties, moieties capable of improving wound healing, moieties capable of influencing the nervous system, moieties having selective affinity for specific tissue or cell types, epitopes and antimicrobial moieties.

In embodiments, a further component or moiety is selected from amino acids, peptides, including cyclic peptides, oligopeptides, polypeptides, glycopeptides and proteins, including glycoproteins; nucleotides, including mononucleotides, oligonucleotides and polynucleotides and carbohydrates. For instance, an amino acid may be linked for stimulating wound healing (L-arginine, L-glutamine) or to modulate the functioning of the nervous system (L-asparagine)

In other embodiments, the further component or moiety is a peptide residue, preferably an oligopeptide residue. Peptides with specific functions are known in the art and may be chosen based upon a known function. For instance, the peptide may be selected from growth factors and other hormonally active peptides. In particular, the moiety may be selected from a peptide residue comprising the sequences as listed in below table. In an embodiment, the moiety is angiotensin. Angiotensin may be used to impart vasoconstriction, increased blood pressure, and/or release of aldosterone from the adrenal cortex.

An example of a suitable peptide is a cyclic peptide like gramicidin S, which is an antimicrobial agent. Further examples of suitable peptides include: vascular endothelial growth factor (VEGF), transforming growth factor B (TGF-B), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), osteogenic protein (OP), monocyte chemoattractant protein (MCP 1), tumor necrosis factor (TNF). Examples of proteins include growth factors, chemokines, cytokines, extracellular matrix proteins, glycosaminoglycans, angiopoetins, ephrins and antibodies. A suitable carbohydrate is heparin, which is antithrombotic. A nucleotide may be selected from therapeutic oligo-nucleotides, such as a oligo-nucleotide for gene therapy and oligo-nucleotide that are capable of binding to cellular or viral proteins, preferably with a high selectivity and/or affinity. Preferred oligo-nucleotides include aptamers. Examples of both DNA and RNA based aptamers are mentioned by Nimjee et. al. (Annu. Rev. Med. 2005, 56, 555-583). The RNA ligand TAR (Trans activation response), which binds to viral TAT proteins or cellular protein cyclin T 1 to inhibit HIV replication, is an example of an aptamer. Further preferred nucleotides include VA-RNA and transcription factor E2F, which regulates cellular proliferation.

The polyfunctional aziridine compound AZ that is used as cross-linking agent in the methods of present disclosure has : a) from 2 to 6 of the following structural units (A): wherein Ri is H;

R and R ₄ are independently chosen from H, a linear group containing from 1 to 8 carbon atoms and optionally containing one or more heteroatoms in the chain;

R ₃ is a linear group containing from 1 to 4 carbon atoms and optionally containing one or more heteroatoms in the chain;

R’= H or an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms;

R” = H, an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms, a cycloaliphatic hydrocarbon group containing from 5 to 12 carbon atoms, an aromatic hydrocarbon group containing from 6 to 12 carbon atoms, CH ₂ -0-(C=0)-R’”, CH ₂ -0-R””, or CH2-(OCR””’HCR””’H)n-OR”””, whereby R’” is an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms and R”” is an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms or an aromatic hydrocarbon group containing from 6 to 12 carbon atoms, n being from 1 to 35, R’”” independently being H or an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms and R””” being an aliphatic hydrocarbon group containing from 1 to 4 carbon atoms; whereby R’ and R” may be part of the same saturated cycloaliphatic hydrocarbon group containing from 5 to 8 carbon atoms; and m is an integer from 1 to 6; b) one or more linking chains wherein each one of these linking chains links two of the structural units (A) present in the polyfunctional aziridine compound; and c) a molar mass in the range of from 600 Da to 5000 Da.

These new polyfunctional aziridine compounds AZ and variations thereof as further discussed hereinafter, are extensively described in patent publication W02020/020714A1 (application PCT/EP2019/069198). For more details on the structural aspects of these aziridine compounds AZ, as well as for their synthesis methods and their properties like genotoxicity, explicit reference is made hereby to this patent publication).

Depending on the targeted use of the cross-linked biopolymer as obtained by present methods, and on requirements for such use with regard to for example solubility and reactivity in the cross-linking method, the skilled person will be able to select a suitable aziridine compound.

In embodiments of present methods, the polyfunctional aziridine compound used can be (or is) dispersed in an aqueous medium, or can be (or is) dissolved in an aqueous medium, optionally by using small amounts of a further component like a (biocompatible) dispersion aid or a good solvent for the compound that is miscible with water as a co-solvent.

In other embodiments, the polyfunctional aziridine compound AZ used in said methods shows good biocompatibility and is not cytotoxic.

In further embodiments, the polyfunctional aziridine compound AZ used in said methods shows no genotoxicity.

In embodiments, the polyfunctional aziridine compound AZ is non-genotoxic and non- mutagenic.

An advantage of using an aziridine compound AZ is that the compound shows reduced toxicity, likely as the -relatively large- molecules cannot easily permeate into a cell. It has been found that the aziridine compounds AZ comprising units (A) as defined above show reduced genotoxicity compared to for example trimethylolpropane tris(2-methyl-1- aziridinepropionate). These polyfunctional aziridine compounds AZ show either only weakly positive induced genotoxicity or even are non-genotoxic, i.e. they show a genotoxicity level comparable with the naturally occurring background. Genotoxicity as reported and as further described in W02020/020714A1 has been measured by the ToxTracker® assay (Toxys, Leiden, the Netherlands). The ToxTracker® assay can be applied for pure substances or for compositions that are the direct products obtained in the preparation of these polyfunctional aziridine compounds.

The term “aliphatic hydrocarbon group” refers to optionally branched alkyl, alkenyl and alkynyl group. The term “cycloaliphatic hydrocarbon group” refers to cycloalkyl and cycloalkenyl group optionally substituted with at least one aliphatic hydrocarbon group. The term “aromatic hydrocarbon group” refers to a benzene ring optionally substituted with at least one aliphatic hydrocarbon group. These optionally substituted aliphatic hydrocarbon groups are preferably alkyl groups. Examples of cycloaliphatic hydrocarbon groups with 7 carbon atoms are cycloheptyl and methyl substituted cyclohexyl. An example of an aromatic hydrocarbon group with 7 carbon atoms is methyl substituted phenyl. Examples of aromatic hydrocarbon groups with 8 carbon atoms are xylyl and ethyl substituted phenyl.

Whilst the structural units (A) present in the polyfunctional aziridine compound AZ may independently have different R , R3, R4, R’, R” and/or m, the structural units (A) present in the polyfunctional aziridine compound are preferably identical to each other.

The polyfunctional aziridine compound AZ comprising units (A) may contain more than one aziridine compound, for example a mixture of polyfunctional aziridine compounds as is obtained when a mixture of polyisocyanates is used as starting material.

The polyfunctional aziridine compound AZ contains from 2 to 6 of the structural units (A), preferably from 2 to 4 and more preferably 2 or 3 structural units (A).

R ₂ and R ₄ are independently chosen from H, a linear group containing from 1 to 8 carbon atoms which optionally contains one or more heteroatoms (preferably selected from N, S and O) in the chain, a branched group containing from 3 to 8 carbon. Preferably, R ₂ and R ₄ are independently chosen from H, an aliphatic hydrocarbon group containing from 1 to 8 carbon atoms. More preferably, R ₂ and R ₄ are independently chosen from H or an aliphatic hydrocarbon group containing from 1 to 4 carbon atoms. More preferably, R ₂ and R ₄ are independently chosen from H or an aliphatic hydrocarbon group containing from 1 to 2 carbon atoms.

R ₃ is a linear group containing from 1 to 4 carbon atoms and optionally containing one or more heteroatoms (preferably selected from N, S and O) in the chain4. R ₃ is preferably an aliphatic hydrocarbon group containing from 1 to 4 carbon atoms. In further embodiments, the aggregate number of carbon atoms in R , R3, R4 together is at most 8, 7, 6, 5, 4, 3, 2 or 1.

In an embodiment, R ₂ is H, R3 is C ₂ Hs and R4 is H. In other embodiments, R ₂ is H, R3 is CH3 and R ₄ is H or CH ₃ ; or R ₂ is H, R ₃ is CH ₃ and R ₄ is H. The (preferred) embodiments for R _2, R ₃ and R ₄ groups as indicated above typically result in a more hydrophilic compound AZ, with better solubility or dispersibility in water.

In embodiments, m is an integer from 1 to 6, preferably m is from 1 to 4, more preferably m is 1 or 2 and most preferably m is 1.

In embodiments, R’ is H or an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms, preferably an alkyl group containing from 1 to 12 carbon atoms. R’ is preferably H or an alkyl group containing from 1 to 4 carbon atoms. More preferably R’ is H or an alkyl group containing from 1 to 2 carbon atoms. Most preferably R’ is H.

In embodiments, R” is H, an aliphatic hydrocarbon group containing from 1 to 8 carbon atoms (preferably from 1 to 4 carbon atoms), a cycloaliphatic hydrocarbon group containing from 5 to 12 carbon atoms, an aromatic hydrocarbon group containing from 6 to 12 carbon atoms, CH ₂ -0-(C=0)-R”\ CH ₂ -0-R””, or CH ₂ -(OCR ^, ””HCR ^, ””H) _n -OR”””, whereby R”’ is an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms and R”” is an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms or an aromatic hydrocarbon group containing from 6 to 12 carbon atoms, n being from 1 to 35, R’”” independently being H or a methyl group and R””” being an aliphatic hydrocarbon group containing from 1 to 4 carbon atoms and preferably an alkyl group with 1 to 4 carbon atoms, or R’ and R” may be part of the same saturated cycloaliphatic hydrocarbon group containing from 5 to 8 carbon atoms. Preferably, R” = H, an aliphatic hydrocarbon group containing from 1 to 4 carbon atoms, CH ₂ - 0-(C=0)-R”\ CH ₂ -0-R””, or CH ₂ -(OCR ^, ””HCR ^, ””H) _n -OR”””, whereby R”’ is an alkyl group containing from 1 to 12 carbon atoms and R”” is an alkyl group containing from 1 to 12 carbon atoms, n being from 1 to 35, preferably 2-30, 3-25 or 4-20, R’”” independently being H or a methyl group and R””” being an alkyl group containing from 1 to 4 carbon atoms; or R’ and R” may be part of the same saturated cycloaliphatic hydrocarbon group containing from 5 to 8 carbon atoms.

In embodiments, R’ is H and R” is an alkyl group containing from 1 to 4 carbon atoms, CH ₂ -0-(C=0)-R”\ CH ₂ -0-R””, or CH ₂ -(OCH ₂ CH ₂ ) _n -OCH ₃ whereby R’” is preferably an alkyl group containing from 3 to 12 carbon atoms, more preferably a branched alkyl group with from 3 to 12 carbon atoms, such as for example neopentyl or neodecyl. Most preferably R’” is a branched C9 alkyl. R”” is preferably an alkyl group containing from 1 to 12 carbon atoms. Nonlimited examples for R”” are ethyl, butyl and 2-ethylhexyl.

In other embodiments, the polyfunctional aziridine compound AZ comprises a further alkyl group containing from 1 to 4 carbon atoms as a substituent on the carbon atom to which R” is attached. Preferably this alkyl group is linear and contains 1 or 2 carbon atoms. More preferably, this substituent is a methyl group.

The molar mass of the polyfunctional aziridine compound AZ comprising units (A) is from 600 to 5000 Da. The molar mass of the polyfunctional aziridine comprising units (A) is preferably at most 3800 Da, or at most 3600, 3000, or 1600 Da. The molar mass of the polyfunctional aziridine compound comprising units (A) is preferably at least 700 Da, or at least 800, 840, or 1000 Da. As used herein, the molar mass of the polyfunctional aziridine compound AZ is the calculated molar mass. The calculated molar mass is obtained by adding the atomic masses of all atoms present in the structural formula of the polyfunctional aziridine compound. If the polyfunctional aziridine compound is a mixture comprising more than one polyfunctional aziridine compound comprising units (A), for example when one or more of the starting materials to prepare the polyfunctional aziridine compound is a mixture, the molar mass calculation can be performed for each compound individually present in the composition. The molar mass of the polyfunctional aziridine compound AZ can also be measured using MALDI- TOF mass spectrometry as described in W02020/020714A1 .

The polyfunctional aziridine compound AZ comprising units (A) comprises one or more linking chains wherein each one of these linking chains links two of the structural units (A). The linking chains present in the polyfunctional aziridine compound preferably consist of from 2 to 300 atoms, more preferably from 5 to 250 and most preferably from 6 to 100 atoms. The atoms of the linking chains are preferably C, N, O, S and/or P, preferably C, N and/or O.

A linking chain is defined as the shortest chain of consecutive atoms that links two structural units (A). The following drawing shows, for an example of a polyfunctional aziridine compound according to the invention, the linking chain between two structural units (A). Any two of the structural units (A) present in the polyfunctional aziridine compound are linked via a linking chain as defined herein. Accordingly, each structural unit (A) present in the polyfunctional aziridine compound is linked to every other structural unit (A) via a linking chain as defined herein. In case the polyfunctional aziridine compound has two structural units (A), the polyfunctional aziridine compound has one such linking chain linking these two structural units.

In case the polyfunctional aziridine compound has three structural units (A), the polyfunctional aziridine compound has three linking chains, whereby each of the three linking chains is linking a structural unit (A) with another structural unit (A), i.e. a first structural unit A is linked with a second structural unit (A) via a linking chain and the first and second structural units (A) are both independently linked with a third structural unit (A) via their respective linking chains.

Polyfunctional aziridine compounds with more than two structural units (A) have a number of linking chains according to the following equation: LC = {(AN-1) x AN)} / 2, wherein LC= the number of linking chains and AN = the number of structural units (A) in the polyfunctional aziridine compound.

If for example, there are 5 structural units (A) in the polyfunctional aziridine compound,

AN = 5; which means that there are {(5-1) x 5} 12 10 linking chains. Preferably, the number of consecutive C atoms and optionally O atoms between the N atom of the urethane group in a structural unit (A) and the next N atom which is either present in the linking chain or which is the N atom of the urethane group of another structural unit A is at most 9, as shown in for example the following polyfunctional aziridine compounds according to the invention:

The polyfunctional aziridine compound comprising units (A) preferably comprises one or more connecting groups wherein each one of these connecting groups connects two of the structural units (A), whereby a connecting group is defined as the array of consecutive functionalities (functionalities as defined herein) connecting two structural units (A). In embodiments, the connecting groups preferably consist of at least one functionality selected from the group consisting of aliphatic hydrocarbon functionality (preferably containing from 1 to 8 carbon atoms), cycloaliphatic hydrocarbon functionality (preferably containing from 4 to 10 carbon atoms), aromatic hydrocarbon functionality (preferably containing from 6 to 12 carbon atoms), isocyanurate functionality, iminooxadiazindione functionality, ether functionality, ester functionality, amide functionality, carbonate functionality, urethane functionality, urea functionality, biuret functionality, allophanate functionality, uretdione functionality and any combination thereof.

The following drawing shows in bold a connecting group for an example of a polyfunctional aziridine compound AZ. In this example, the connecting group connecting two of the structural units (A) consists of the array of the following consecutive functionalities: aliphatic hydrocarbon functionality 1 (a linear C ₆ HI ), isocyanurate 2 (a cyclic C3N3O3) functionality and aliphatic hydrocarbon functionality 3 (a linear C ₆ HI ₂ ).

The following drawing shows in bold the connecting group for the following example of a polyfunctional aziridine compound comprising units (A). In this example, the connecting group connecting the two structural units (A) consists of the array of the following consecutive functionalities: aliphatic hydrocarbon functionality 1 (a linear C ₆ HI ), isocyanurate 2 (a cyclic C3N3O3) and aliphatic hydrocarbon functionality 3 (a linear C6H12).

Any two of the structural units (A) present in the polyfunctional aziridine compound are connected via a connecting group as defined herein. Accordingly, each structural unit (A) present in the polyfunctional aziridine compound AZ is connected to every other structural unit (A) with a connecting group as defined in the invention. In case the polyfunctional aziridine compound has two structural units (A), the polyfunctional aziridine compound has one such connecting group connecting these two structural units. In case the polyfunctional aziridine compound has three structural units (A), the polyfunctional aziridine compound has three such connecting groups, whereby each one of the three connecting groups is connecting a structural unit (A) with another structural unit (A).

The following drawing shows for an example of a polyfunctional aziridine compound having three structural units (A), the three connecting groups whereby each one of the three connecting groups is connecting two structural units (A). One connecting group consists of the array of the following consecutive functionalities: aliphatic hydrocarbon functionality 1 (a linear C ₆ HI ), isocyanurate 2 (a cyclic C3N3O3) and aliphatic hydrocarbon functionality 3 (a linear C ₆ HI ₂ ) connecting the structural units (A) which are labelled as A1 and A2. For the connection between structural units (A) which are labelled as A1 and A3 , the connecting group consists of the array of the following consecutive functionalities: aliphatic hydrocarbon functionality 1 (a linear C ₆ HI ₂ ), isocyanurate 2 (a cyclic C3N3O3) and aliphatic hydrocarbon functionality 4 (a linear C ₆ HI ₂ ), while for the connection between the structural units (A) which are labelled as A2 and A3, the connecting group consists of the array of the following consecutive functionalities: aliphatic hydrocarbon functionality 3 (a linear ObHi ₂ ), isocyanurate 2 (a cyclic C3N3O3) and aliphatic hydrocarbon functionality 4 (a linear C ₆ HI ₂ ) . The following drawing shows, another example of a polyfunctional aziridine compound, with the linking chain between two structural units (A).

In this example, the connecting group connecting the two structural units (A) consists of the array of the following consecutive functionalities: aliphatic hydrocarbon functionality 1 (a branched C ₃ H ₆ ), aromatic hydrocarbon functionality 2 (a benzene ring) and aliphatic hydrocarbon functionality 3 (a branched C ₃ H ₆ ).

In another example of the polyfunctional aziridine compound AZ, the connecting group connecting the two structural units (A) consists of the array of the following consecutive functionalities: aliphatic hydrocarbon functionality 1 (a linear C ₆ H ₁₂ ), uretdione 2 (a cyclic C ₂ N ₂ O ₂ ) and aliphatic hydrocarbon functionality 3 (a linear C ₆ HI ). Preferably, the connecting groups consist of at least one functionality selected from the group consisting of aliphatic hydrocarbon functionality (preferably containing from 1 to 8 carbon atoms), cycloaliphatic hydrocarbon functionality (preferably containing from 4 to 10 carbon atoms), aromatic hydrocarbon functionality (preferably containing from 6 to 12 carbon atoms), isocyanurate functionality, iminooxadiazindione functionality, urethane functionality, urea functionality, biuret functionality and any combination thereof. The connecting groups preferably contain an isocyanurate functionality, an iminooxadiazindione functionality, a biuret functionality, allophanate functionality or an uretdione functionality. More preferably, the connecting groups contain an isocyanurate functionality or an iminooxadiazindione functionality. For the sake of clarity, the polyfunctional aziridine compound may be obtained from the reaction product of one or more suitable compound B and a hybrid isocyanurate such as for example a HDI/IPDI isocyanurate, resulting in a polyfunctional aziridine compound with a connecting group consisting of the array of the following consecutive functionalities: a linear C ₆ Hi (i.e. an aliphatic hydrocarbon functionality with 6 carbon atoms), an isocyanurate functionality (a cyclic C3N3O3) and

(i.e. a cycloaliphatic hydrocarbon functionality with 9 carbon atoms and an aliphatic hydrocarbon functionality with 1 carbon atom).

The term “aliphatic hydrocarbon functionality” refers to optionally branched alkyl, alkenyl and alkynyl groups. Whilst the optional branches of C atoms are part of the connecting group, they are not part of the linking chain. The term “cycloaliphatic hydrocarbon functionality” refers to cycloalkyl and cycloalkenyl groups optionally substituted with at least one aliphatic hydrocarbon group. Whilst the optional aliphatic hydrocarbon group substituents are part of the connecting group, they are not part of the linking chain. The term “aromatic hydrocarbon functionality” refers to a benzene ring optionally substituted with at least one aliphatic hydrocarbon group. The optionally substituted aliphatic hydrocarbon group is preferably an alkyl group. Whilst the optional aliphatic hydrocarbon group substituents are part of the connecting group, they are not part of the linking chain. On the connecting groups, one or more substituents may be present as pendant groups on the connection group, as shown in bold in for example the following polyfunctional aziridine compound. These pendant groups are not part of the connecting groups. Such pending groups may enhance solubility of the compound in aqueous media, like an oligomer of ethylene oxide, vinyl pyrrolidone or an oxazoline. Especially when the pendant groups contain ethylene oxide units, as exemplified in below drawing, the compound shows better water solubility. Fii

An aziridinyl group has the following structural formula: 14

AAA An isocyanurate functionality is defined as

A w _, t

Y ^M Y

An iminooxadiazindione functionality is defined as O O

An allophanate functionality is defined as

O

An uretdione functionality is defined as A biuret functionality is defined as

In an embodiment, the connecting groups present in the polyfunctional aziridine compound comprising units (A) consist of the following functionalities: at least one aliphatic hydrocarbon functionality and/or at least one cycloaliphatic hydrocarbon functionality and optionally at least one aromatic hydrocarbon functionality and optionally an isocyanurate functionality or iminooxadiazindione functionality or allophanate functionality or uretdione functionality. Preferably, the connecting groups present in the polyfunctional aziridine compound AZ consist of the following functionalities: at least one aliphatic hydrocarbon functionality and/or at least one cycloaliphatic hydrocarbon functionality and optionally at least one aromatic hydrocarbon functionality and optionally an isocyanurate functionality or iminooxadiazindione functionality. A very suitable way of obtaining such polyfunctional aziridine compound is reacting a compound B having the following structural formula: with a polyisocyanate with aliphatic reactivity. The term “a polyisocyanate with aliphatic reactivity” being intended to mean compounds in which all of the isocyanate groups are directly bonded to aliphatic or cycloaliphatic hydrocarbon groups, irrespective of whether aromatic hydrocarbon groups are also present. The polyisocyanate with aliphatic reactivity can be a mixture of polyisocyanates with aliphatic reactivity. Compounds based on polyisocyanate with aliphatic reactivity have a reduced tendency of yellowing overtime when compared to a similar compound but based on polyisocyanate with aromatic reactivity. The term “a polyisocyanate with aromatic reactivity” being intended to mean compounds wherein all of the isocyanate groups are directly bonded to aromatic hydrocarbon groups, irrespective of whether aliphatic or cycloaliphatic groups are also present. Preferred polyisocyanates with aliphatic reactivity are 1 ,5-pentamethylene diisocyanate (PDI), 1 ,6-hexamethylene diisocyanate (HDI), isophorone diisocyanate (IPDI), 4,4'-dicyclohexyl methane diisocyanate (H12MDI), 2,2,4-trimethyl hexamethylene diisocyanate, 2,4,4-trimethyl hexamethylene diisocyanate, tetramethylxylene diisocyanate (TMXDI, all isomers) and higher molar mass variants like for example their isocyanurates, or iminooxadiazindiones. In this embodiment, preferably the connecting groups consist of the array of the following consecutive functionalities: aliphatic hydrocarbon functionality, aromatic hydrocarbon functionality and aliphatic hydrocarbon functionality (for example when using TMXDI for preparing the polyfunctional aziridine compound) or the connecting groups consist of the array of the following consecutive functionalities: cycloaliphatic hydrocarbon functionality, aliphatic hydrocarbon functionality and cycloaliphatic hydrocarbon functionality (for example when using H12MDI for preparing the polyfunctional aziridine compound) or more preferably, the connecting groups consist of the array of the following consecutive functionalities: aliphatic hydrocarbon functionality, isocyanurate functionality or iminooxadiazindione functionality, and aliphatic hydrocarbon functionality. Most preferably, in this embodiment, the connecting group consists of the array of the following consecutive functionalities: aliphatic hydrocarbon functionality, isocyanurate functionality, and aliphatic hydrocarbon functionality (for example when using an isocyanurate of 1 ,6-hexamethylene diisocyanate and/or an isocyanurate of 1 ,5-pentamethylene diisocyanate for preparing the polyfunctional aziridine compound).

In another embodiment, the polyfunctional aziridine compound AZ is according to the following structural formula: wherein Z is a molecular residue obtained by removing isocyanate reactive groups XH of a molecule; q is an integer from 2 to 6; i is the index for the different groups D and is an integer from 1 to q;

Di groups independently have the following structural formula wherein X is NRn, S or O, whereby Rn is H or an alkyl group with 1 to 4 carbon atoms; Y is an aromatic hydrocarbon group, an aliphatic hydrocarbon group, a cycloaliphatic hydrocarbon group or a combination thereof; j is an integer from 1 to p; p is an integer from 0 to 10; and m, R', R", Ri, R , R3 and R ₄ are as defined above. In this embodiment, the polyfunctional aziridine compound contains from 2 to 6 D, groups. Whilst the structural units D, may independently be the same or different, the structural units D, are preferably identical to each other.

Isocyanate reactive groups XH are herein defined as hydroxy groups (X is O), primary (X is NH) or secondary amines (X is NRn in which Rn is an alkyl group with 1 to 4 carbon atoms) or mercaptans (X is S). Preferred isocyanate reactive groups XH are hydroxy groups (X is O), primary amines (X is NH) or secondary amines (X is NRn in which Rn is an alkyl group with 1 to 4 carbon atoms). More preferred isocyanate reactive groups XH are hydroxy groups (X is O) and primary amines (X is NH). The molecule from which isocyanate reactive group are removed to obtain Z is preferably a diol, a triol, a polyether with terminal isocyanate reactive groups, a polyamide with terminal isocyanate reactive groups, a polycarbonate with terminal isocyanate reactive groups, or a polysiloxane with terminal isocyanate reactive groups which groups are linked to the siloxane via at least one carbon atom. In case Z is a molecular residue obtained by removing isocyanate reactive groups XH of a diol or a triol, the isocyanate reactive groups XH are hydroxy groups and thus X is O. In case Z is a molecular residue obtained by removing isocyanate reactive groups XH of a polyether with terminal isocyanate reactive groups or of a polyamide with terminal isocyanate reactive groups, the isocyanate reactive groups XH are preferably NH2 (thus X is NH) or OH (thus X is O) and more preferably the isocyanate reactive groups XH are OH (thus X is O). In case Z is a molecular residue obtained by removing isocyanate reactive groups XH of a polycarbonate with terminal isocyanate reactive groups, the isocyanate reactive groups are preferably OH and thus X is O.

In case j is larger than 1 , Z can be the same or different.

Preferably, q is 2 or 3 and more preferably, q is 1.

Preferably, p is an integer from 0 to 10, more preferably from 0 to 5, most preferably from O to 3.

In this embodiment, p is most preferably 0 for all D, and accordingly D, independently have the following structural formula wherein X, Y, m, R', R", Ri, R ₂ , R3 and R ₄ are as defined above. Preferably m is 1.

Whilst the structural units D, may independently be the same or different, the structural units Di are preferably identical to each other.

The total amount of cyclic structures (apart from the aziridine groups) present in the polyfunctional aziridine compound AZ is preferably at most 3, since this results in a lower viscosity than when a higher amount of cyclic structures is present. Lower viscosity is easier to handle and/or less co-solvent is needed to make the compound more easy to handle. A polyfunctional aziridine compound with more than three cyclic structures may result in more difficulties when dissolving such polyfunctional aziridine if the polyfunctional aziridine compound is solid at ambient temperature. The total amount of cyclic structures (apart from the aziridine groups) present in the polyfunctional aziridine compound is more preferably from 0 to 2, even more preferably is 1 or 2, and most preferably is 1 , which is preferably an isocyanurate or an iminooxadiazindione.

The polyfunctional aziridine compound preferably contains at least 5 mass%, more preferably at least 5.5, 6, 9 or at least 12 mass %, and preferably less than 25 or less than 20 mass % of urethane bonds. The polyfunctional aziridine compound AZ preferably has an aziridine equivalent mass (molar mass of the polyfunctional aziridine compound divided by number of aziridinyl groups present in the polyfunctional aziridine compound) of at least 200, more preferably at least 230 and even more preferably at least 260 Daltons and preferably at most 2500, more preferably at most 1000 and even more preferably at most 500 Da.

The polyfunctional aziridine compound AZ is preferably obtained by reacting at least a polyisocyanate and a compound B with the following structural formula: whereby the molar ratio of compound B to polyisocyanate is from 2 to 6, more preferably from 2 to 4 and most preferably from 2 to 3, and whereby m, R', R", Ri, R , R3 and R ₄ are as defined above. For more details on such synthesis methods, explicit reference is hereby made to W02020/020714A1 (PCT/EP2019/069198) .

The amount of alkoxy poly(ethyleneglycol), preferably methoxy poly(ethyleneglycol) (MPEG), and/or poly(ethyleneglycol) (PEG) chains with a number average molar mass M _n higher than 1600 Da, preferably with a M _n higher than 2200 Da in the polyfunctional aziridine compound as defined above is preferably less than 35 mass %, more preferably less than 15 mass %, and preferably more than 1 mass % or more than 2, 3, 4, or 5 mass %; to obtain a compound soluble or dispersible in aqueous medium. The methoxy poly(ethyleneglycol)

(MPEG) and/or poly(ethyleneglycol) (PEG) chains present in the polyfunctional aziridine compound AZ preferably have a M _n lower than 1600 Da, or lower than 100, 770 or 570 Da.

Other examples of polyfunctional aziridine compounds comprising units (A) that may be used include , and

Another example of a suitable polyfunctional aziridine compound AZ is represented by the following structural formula:

Such compound comprises at least one ethylene oxide oligomer as R”, preferably 2, 3 or an average of from 2 to 3 ethylene oxide oligomers as R”. Preferably, each oligomer independently contains at most 35, 30, 25 , 20 or 15 units and at least 2, 3, 4 or 5 ethylene oxide units; or a molar mass at least 85, 125, 170, 210 Da and at most 1500, 1300, 1100, 900 or 700 Da, like about 600 Da. These compounds show advantageously increased hydrophilic character and enhanced solubility in water; enabling efficient reaction with proteins in aqueous medium. The molar mass of the polyfunctional aziridine compound is in the range of from 600 to

5000 Da. Molar mass be calculated from the structural formula or determined as the number average molar mass with known methods. Preferred molar masses are as described above and molar mass of the polyfunctional aziridine compounds may be determined using MALDI-TOF- MS as described in W02020/020714A1. MALDI-TOF-MS means matrix-assisted laser desorption ionization time of flight mass spectroscopy.

The average number of aziridinyl groups in the polyfunctional aziridine cross-linker AZ is preferably at least 1.8, more preferably at least 2 or 2.2, and preferably less than 10, more preferably less than 6 or 4. Most preferably, the average number of aziridinyl groups is from 2.2 to 3. The calculated average amount of urethane bonds is at least 5 mass %, more preferably at least 5.5. 6, 9, or 12 mass % and preferably less than 25 or 20 mass % of urethane bonds, relative to the total mass of the polyfunctional aziridine compounds AZ.

The polyfunctional aziridine compounds AZ preferably have a Brookfield viscosity of at least 500 mPa.s at 25 °C, more preferably at least 1200 or 3000 mPa.s at 25 °C and preferably at most 1000000, more preferably at most 100000, 30000, 10000 or at most 5000 mPa.s at 25 °C. As used herein, the Brookfield viscosity is determined according to ISO 2555-89. In an alternative embodiment the viscosity of the polyfunctional aziridine was measured with a Brookfield with spindle S63, at 25 °C at 80% solids, 20% in dimethyl formamide (DMF). The viscosity as measured according to this method is preferably in the range of 300 to 20000 mPas, more preferably in the range of from 500 to 12000 and most preferably in the range of from 700 to 3000 mPas.

The polyfunctional aziridine compounds AZ can be advantageously used as cross-linking agent for cross-linking a carboxylic acid functional biopolymer dissolved and/or dispersed in a solvent, preferably in an aqueous medium.

In embodiments, the invention provides a method of making an article comprising a cross-linked biopolymer, the method comprising steps of forming a biopolymer material or biopolymer composition into an article of a desired shape, adding a polyfunctional aziridine compound AZ to the biopolymer before, during and/or after the forming step, and reacting the biopolymer with the polyfunctional aziridine compound to make an article comprising the cross- linked biopolymer. Such steps can be performed using solutions or dispersions, preferably aqueous solutions or dispersions of biopolymer and of cross-linker, depending on the article to be made; which article may for example be a 3-dimensional scaffold structure or a thin coating layer on a substrate. Cross-linking an aqueous solution of hydrophilic biopolymer may typically result in a hydrogel, that is a cross-linked biopolymer network swollen with water. A solid porous structure can subsequently be made by removing the water; for example, by a freeze drying process.

In further embodiments, the disclosure for example provides a method of making a coating comprising a cross-linked biopolymer on a substrate, the method comprising steps of

• Making a solution or dispersion of a biopolymer material or a biopolymer, preferably in an aqueous medium; • Mixing a polyfunctional aziridine compound AZ into the solution or dispersion comprising biopolymer;

• Applying the obtained mixture to a surface of the substrate to form a coating;

• Drying the formed coating, and

• Cross-linking the biopolymer in the coating before, during and/or after drying.

In embodiments, the step of making a solution or dispersion of biopolymer may comprise dissolving and/or dispersing the biopolymer (material) in an aqueous medium. Depending on the type of biopolymer material and whether it comprises polymeric components with a specific structural conformation that would need to be preserved, the skilled person will be able to select, optionally based on some experiments, suitable conditions like temperature, time, pH, auxiliary equipment like a stirrer, and optionally additional components like a dispersion agent or a co-solvent to make a solution or dispersion of the biopolymer.

The step of mixing a polyfunctional aziridine compound AZ into the solution or dispersion may comprise adding the compound as a pure material in liquid or solid form, but also as an aqueous dispersion or as a solution in a solvent. Like for the previous step, the skilled person will be able to select a suitable way and conditions to add the aziridine compound. Preferably, the solution or dispersion of biopolymer and the aziridine compound or aziridine compound solution or dispersion are separately stored, for example at ambient conditions, since the reaction between the cross-linking agent and the biopolymer to be cross-linked may start immediately after mixing the (aqueous) compositions of biopolymer and aziridine compound. For the same reason, mixing the polyfunctional aziridine compound into the solution or dispersion of biopolymer may preferably be done just prior to the subsequent step of forming a coating. The composition obtained by mixing the aziridine compound into the solution or dispersion of biopolymer may comprise these two components in such amounts, that the ratio of aziridinyl groups of the aziridine compound to (pendant or end) functional groups of the biopolymer, like carboxylic acid groups, is from 0.01 to 5.0, preferably at least 0.05, 0.1 , 0.2, 0.25, or 0.3 and at most 4, 3, 2, 1.5 1.0, 0.95, 0.9, 0.85 or 0.8; like from 0.2 to 1.5, from 0.25 to 0.95, or from 0.3 to 0.8. An advantage of an excess of functional groups on the biopolymer relative to aziridinyl groups may be that all such latter reactive groups will likely be reacted.

The step of applying the obtained mixture to a surface of a substrate to form a coating may be done using well-known application methods, including for example casting, brushing, dipping or spraying. Again, the skilled person will be able to select a suitable method and conditions, depending on for example the type of substrate, the surface of the substrate to be coated, starting viscosity of the mixture, and time until the cross-linking reaction results in such increase in viscosity of the mixture that it would hamper proper coating. The mixture can be applied to a specific part of the surface of a substrate, but also to all the surface of the substrate. The type of substrate can also vary widely regarding its physical form and material at its surface. The substrate may be solid and non-porous; or can be porous or fibrous. The substrate can be a medical device or implant, and be made from natural or synthetic materials, including metals, ceramics, and organic polymers, as is known in the art. Suitable metals include titanium and stainless-steel grades, like surgical steel. Ceramics include calcium phosphates, like hydroxyapatite, TCP, bioglass, and mixtures thereof. Suitable polymer substrates include bioinert polymers like PEEK, UHMWPE, and biodegradable polymers like copolymers based on lactic acid, polyhydroxyesters, and polyesteramide copolymers, like such copolymers comprising natural amino acids.

The step of drying the formed coating layer comprises removing solvent and/or other volatiles, like water and optionally co-solvent, for example by evaporation under mild conditions of temperature and time, and optionally under reduced pressure. The skilled person will be able to select suitable conditions, depending for example on stability of the biopolymer, and whether its structural conformation is to be maintained. In embodiments, the step comprises freezedrying the coating.

The step of cross-linking, that is reacting the functional groups of the biopolymer with the aziridinyl groups of the aziridine compound, may be effected before, during and/or after drying. The skilled person will be aware that the rate at which the cross-linking reaction proceeds is dependent on reactivity of the functional groups concerned, on temperature, and on optional presence of compounds that may affect said reaction. The reaction may be accelerated by exposing the coating to an energy source, like heat or light; or may be slowed down by cooling, for example by applying a freeze-drying process. In embodiments, cross-linking mainly occurs during and/or after drying of the coating, preferably after drying.

The thickness of the dried and cured coating layer thus formed on the substrate may vary widely. Depending on the targeted application, the thickness can for example be from 0.1 to 200 pm. In embodiments, coating thickness is at least 0.5, 1 , 2, 5 or 10 pm and at most 150, 100, 50 or 25 pm.

In other embodiments, the cross-linking method or the coating method may be repeated one or multiple times; for example as steps of an additive manufacturing process or 3D-printing process, wherein a multi-layer coating or a 3-dimensional article is formed layer-by-layer.

In a further aspect, the disclosure provides an article or a component comprising cross- linked biopolymer as obtainable by or as obtained with the above methods, including a 3D- printing process comprising steps according to the cross-linking or coating method. Examples of such article or component include a porous collagen-based sheet or other structure for use in for example tissue engineering, or (a component of) an orthopedic or dental implant having a collagen-based coating on at least part of its surface.

In other aspects this disclosure provides a medical device or medical implant, comprising an article or a component comprising cross-linked biopolymer (material) as obtainable by or as obtained with the above methods.

In embodiments, the medical device or medical implant comprises on at least part of its surface a coating comprising cross-linked biopolymer (material) as obtainable by or as obtained with the above methods. The medical device or implant may have been made from different materials, including metals and organic polymers, and may have different physical shapes; including non-porous, monolithic implants like a titanium screw, and porous structures like fibrous constructs such as sutures or fabrics comprising polyester or polyethylene yarns.

The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising”, “having”, “including” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., “such as” or “like”) provided herein, is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. While certain optional features are described as embodiments of the invention, the description is meant to encompass and specifically disclose all combinations of these embodiments unless specifically indicated otherwise or physically impossible.

The various embodiments and ways of performing aspects of the invention as described above, are hereafter further summarized by a series of exemplary embodiments. [1] A method of cross-linking a biomaterial containing a natural or a synthetic biopolymer, the method comprising a step of reacting functional groups of the biopolymer with aziridine (or aziridinyl) groups of a polyfunctional aziridine compound AZ as cross-linking agent, which compound has: a) from 2 to 6 of the following structural units (A): wherein Ri is H;

R and R ₄ are independently chosen from H, a linear group containing from 1 to 8 carbon atoms and optionally containing one or more heteroatoms in the chain;

R ₃ is a linear group containing from 1 to 4 carbon atoms and optionally containing one or more heteroatoms in the chain;

R’= H or an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms;

R” = H, an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms, a cycloaliphatic hydrocarbon group containing from 5 to 12 carbon atoms, an aromatic hydrocarbon group containing from 6 to 12 carbon atoms, CH ₂ -0-(C=0)-R’”, CH ₂ -0-R””, or CH ₂ -(OCR””’HCR””’H)n-OR”””, whereby R’” is an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms and R”” is an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms or an aromatic hydrocarbon group containing from 6 to 12 carbon atoms, n being from 1 to 35, R’”” independently being H or an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms and R””” being an aliphatic hydrocarbon group containing from 1 to 4 carbon atoms; whereby R’ and R” may be part of the same saturated cycloaliphatic hydrocarbon group containing from 5 to 8 carbon atoms; and m is an integer from 1 to 6; b) one or more linking chains wherein each one of these linking chains links two of the structural units (A) present in the polyfunctional aziridine compound; and c) a molar mass in the range of from 600 Da to 5000 Da.

[2] A method of making an article comprising a cross-linked biopolymer, the method comprising steps of forming a composition comprising biopolymer into an article of a desired shape, adding a polyfunctional aziridine compound AZ to the composition before, during and/or after the forming step, and cross-linking the biopolymer according to embodiment [1] to make an article comprising the cross-linked biopolymer. [3] A method of making a coating comprising a cross-linked biopolymer on a substrate, the method comprising steps of

• Making a solution or dispersion of a biopolymer material, for example an aqueous solution or dispersion;

• Mixing a polyfunctional aziridine compound AZ into the solution or dispersion;

• Applying the obtained mixture to a surface of the substrate to form a coating;

• Drying the formed coating, and

• Cross-linking the biopolymer in the coating before, during and/or after drying according to embodiment [1]

[4] The method of any one of embodiments 1-3, wherein the biopolymer (material) is provided as a solution or dispersion of a hydrophilic biopolymer in an aqueous medium to result in a hydrogel containing cross-linked biopolymer.

[5] The method of any one of embodiments 1-4, wherein the steps are repeated multiple times to make a multi-layer coating or a 3-dimensional article.

[6] The method of any one of embodiments 1-5, wherein functional groups of the biopolymer react with the polyfunctional aziridine compound to form cross-links and with functional groups present on a surface of a substrate to result in (cross-linked) biopolymer being chemically bound to the surface.

[7] The method of any one of embodiments 1-6, wherein the biopolymer in the biopolymer material has a molar mass of 200-2.000.000 g/mol, and can be an oligomer with molar mass of about 200-10.000 g/mol or 300-5.000 g/mol, or a polymer with molar mass of at least 5.000, 10.000, 15.000 or 20.000 g/mol and at most 1.000.000 or 500.000 g/mol.

[8] The method of any one of embodiments 1-7, wherein the biopolymer material comprises a natural biopolymer.

[9] The method of any one of embodiments 1-8, wherein the biopolymer material substantially consists of a natural biopolymer.

[10] The method of any one of embodiments 1 -9, wherein the biopolymer material comprises a protein, a polynucleotide, a polysaccharide, or a bacterial polyester.

[11] The method of any one of embodiments 1-10, wherein the biopolymer material comprises a polypeptide, like a high molar mass protein.

[12] The method of any one of embodiments 1-11 , wherein the biopolymer material comprises a collagen having a fibrous structure based on aggregated fibrils formed from three helically-ordered peptide chains. [13] The method of any one of embodiments 1-10, wherein the biopolymer material comprises a protein of relatively low mass, like a peptide or protein fragment.

[14] The method of any one of embodiments 1-10, wherein the biopolymer material comprises a polynucleotide like DNA or RNA

[15] The method of any one of embodiments 1-10, wherein the biopolymer material comprises an oligomeric or polymeric polysaccharide like a cellulose, starch, heparin, alginate, carrageenan, chitin, chitosan, or a derivative thereof.

[16] The method of any one of embodiments 1-7, wherein the biopolymer material comprises or substantially consists of a synthetic biopolymer, like a (co)polymer of lactic acid and glycolic acid, or a polyhydroxybutyrate.

[17] The method of any one of embodiments 1-13, wherein the biopolymer material comprises a collagen, like a fibrous collagen, an acid-soluble collagen, or a mixture of different collagens.

[18] The method of any one of embodiments 1-13, wherein the biopolymer material comprises or substantially consists of a fibrous collagen.

[19] The method of any one of embodiments 1-13, wherein the biopolymer material comprises or substantially consists of a mixture of one or more fibrous collagen types and a soluble collagen or elastin.

[20] The method of any one of embodiments 1-19, wherein the biopolymer material comprises a biopolymer having one or more, preferably two or more nucleophilic groups as functional groups, which groups may be pendant groups and/or end groups.

[21] The method of embodiment 20, wherein the biopolymer comprises at least one hydroxyl, amine, thiol, sulphonic acid, or carboxyl group, preferably at least 2, 4, 6, or 8 nucleophilic groups.

[22] The method of any one of embodiments 19-21 , wherein the biopolymer comprises at least one carboxyl group, preferably at least 2, 4, 6, or 8 groups.

[23] The method of any one of embodiments 1-22, wherein the reaction of functional groups of the biopolymer with the aziridine-functional compound is performed in an aqueous environment and at a temperatures of between 0 and 80 °C, or between 0 and 35 °C.

[24] The method of any one of embodiments 1-23, wherein the biopolymer material contains at least one further component in addition to a biopolymer. [25] The method of embodiment 24, wherein the biopolymer material comprises a collagen as biopolymer and ECM components as further components.

[26] The method of embodiment 24, wherein the further components include at least one organic compound such as a processing aid, a bioactive agent like an antimicrobial or antibacterial agent, and/or inorganic particles like bioceramic particles like calcium phosphate based ceramics such as hydroxyapatite.

[27] The method of any one of embodiments 24-26, wherein the further components include a cell signaling moiety, a moiety capable of improving cell adhesion, a moiety capable of controlling cell growth (such as stimulation or suppression of proliferation), an antithrombotic moiety, a moiety capable of improving wound healing, a moiety capable of influencing the nervous system, a moiety having selective affinity for specific tissue or cell types, epitopes, and/or an antimicrobial moiety.

[28] The method of any one of embodiments 1-27, wherein the the biopolymer material comprises a biopolymer and from 0.1 to 10 mass% of organic further components, preferably at least 0.2, 0.4, 0.6, 0.8, 1 or 2 mass% and at most 9, 8, 7, 6, 5, 4, or 3 mass% of further components (based on total mass of biopolymer and further components); or the solution or dispersion of biopolymer material or the biopolymer composition comprises biopolymer and from about 5 to about 90 mass% of inorganic particles like bioceramic particles such as hydroxyapatite (based on total mass of biopolymer and further components), preferably at least 10, 15, 20, or 25 mass% and at most 85, 80, 75, 70, 65, 60, 55 or 50 mass%.

[29] The method of any one of embodiments 1-28, wherein the polyfunctional aziridine compound AZ is a compound having a structure and properties as described in patent publication W02020/020714A1.

[30] The method of any one of embodiments 1-29, wherein the polyfunctional aziridine compound AZ is dispersed or dissolved in an aqueous medium, optionally by using small amounts of a further compound like a dispersion aid or a good solvent for compound AZ.

[31] The method of any one of embodiments 1-30, wherein the polyfunctional aziridine compound AZ shows good biocompatibility, and limited or no cytotoxicity or genotoxicity.

[32] The method of any one of embodiments 1-31, wherein the polyfunctional aziridine compound AZ has structural units (A) wherein R ₂ , R3, R ₄ , R’, R” and m are identical in each unit (A).

[33] The method of any one of embodiments 1-32, wherein the polyfunctional aziridine compound AZ contains more than one aziridine compound, for example a mixture of polyfunctional aziridine compounds that have been obtained by a synthesis method using a mixture of polyisocyanates as starting material.

[34] The method of any one of embodiments 1-33, wherein the polyfunctional aziridine compound AZ contains from 2 to 4, preferably 2 or 3 structural units (A).

[35] The method of any one of embodiments 1-34, wherein R and R ₄ of the polyfunctional aziridine compound AZ are independently chosen from H, a linear group containing from 1 to 8 carbon atoms which optionally contains one or more heteroatoms (preferably selected from N, S and O) in the chain, or a branched group containing from 3 to 8 carbon.

[36] The method of any one of embodiments 1 -34, wherein R ₂ and R ₄ of the polyfunctional aziridine compound AZ are independently chosen from H and an aliphatic hydrocarbon group containing from 1 to 8 carbon atoms, preferably, R ₂ and R ₄ are independently chosen from H and an aliphatic hydrocarbon group containing from 1 to 4 carbon atoms, or R ₂ and R ₄ are independently chosen from H and an aliphatic hydrocarbon group containing from 1 to 2 carbon atoms.

[37] The method of any one of embodiments 1-36, wherein R ₃ of the polyfunctional aziridine compound AZ is a linear group containing from 1 to 4 carbon atoms and optionally containing one or more heteroatoms (preferably selected from N, S and O) in the chain, preferably R ₃ is an aliphatic hydrocarbon group containing from 1 to 4 carbon atoms.

[38] The method of any one of embodiments 1-37, wherein the aggregate number of carbon atoms in R ₂ , R ₃ , R ₄ is at most 8, 7, 6, 5, 4, 3, 2 or 1.

[39] The method of any one of embodiments 1-38, wherein R ₂ is H, R ₃ is C ₂ Hs and R ₄ is H; or R ₂ is H, R ₃ is CH ₃ and R ₄ is H or CH ₃ ; or R ₂ is H, R ₃ is CH ₃ and R ₄ is H.

[40] The method of any one of embodiments 1-39, wherein m is an integer from 1 to 6, preferably m is from 1 to 4, m is 1 or 2, or m is 1.

[41] The method of any one of embodiments 1-40, wherein R’ is H or an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms, preferably R’ is H or an alkyl group containing from 1 to 12 carbon atoms, H or an alkyl group containing from 1 to 4 carbon atoms, H or an alkyl group containing from 1 to 2 carbon atoms, or R’ is H.

[42] The method of any one of embodiments 1-41, wherein R” is H, an aliphatic hydrocarbon group containing from 1 to 8 carbon atoms (preferably from 1 to 4 carbon atoms), a cycloaliphatic hydrocarbon group containing from 5 to 12 carbon atoms, an aromatic hydrocarbon group containing from 6 to 12 carbon atoms, CH ₂ -0-(C=0)-R’”, CH ₂ -0-R””, or CH ₂ -(OCR””’HCR””’H)n-OR”””, wherein R’” is an aliphatic hydrocarbon group containing from

1 to 12 carbon atoms and R”” is an aliphatic hydrocarbon group containing from 1 to 12 carbon atoms or an aromatic hydrocarbon group containing from 6 to 12 carbon atoms, n being from 1 to 35, R’”” independently being H or a methyl group and R””” being an aliphatic hydrocarbon group containing from 1 to 4 carbon atoms and preferably an alkyl group with 1 to 4 carbon atoms; or R’ and R” may be part of the same saturated cycloaliphatic hydrocarbon group containing from 5 to 8 carbon atoms.

[43] The method of any one of embodiments 1-41, wherein R” is H, an aliphatic hydrocarbon group containing from 1 to 4 carbon atoms, CH -0-(C=0)-R’”, CH ₂ -0-R””, or CH ₂ - (OCR””’HCR””’H)n-OR”””, wherein R’” is an alkyl group containing from 1 to 12 carbon atoms and R”” is an alkyl group containing from 1 to 12 carbon atoms, n being from 1 to 35, preferably 2-30, 3-25 or 4-20, R’”” independently being H or a methyl group, and R””” being an alkyl group containing from 1 to 4 carbon atoms; or R’ and R” may be part of the same saturated cycloaliphatic hydrocarbon group containing from 5 to 8 carbon atoms.

[44] The method of any one of embodiments 1-43, wherein R’ is H; R” is an alkyl group containing from 1 to 4 carbon atoms, CH ₂ -0-(C=0)-R’”, CH ₂ -0-R””, or CH ₂ -(OCH ₂ CH ₂ ) _n -OCH3, and wherein R’” is preferably an alkyl group containing from 3 to 12 carbon atoms, a branched alkyl group with from 3 to 12 carbon atoms such as for example neopentyl or neodecyl, or a branched C9 alkyl, and R”” is preferably an alkyl group containing from 1 to 12 carbon atoms such as ethyl, butyl or2-ethylhexyl.

[45] The method of any one of embodiments 1-44, wherein, the polyfunctional aziridine compound AZ comprises a further alkyl group containing from 1 to 4 carbon atoms as a substituent on the carbon atom to which R” is attached; preferably this alkyl group is linear and contains 1 or 2 carbon atoms, or this alkyl substituent is a methyl group.

[46] The method of any one of embodiments 1-45, wherein the polyfunctional aziridine compound AZ has a calculated (average) molar mass of at most 3800 Da, or at most 3600, 3000, or 1600 Da; and of at least 700 Da, or at least 800, 840, or 1000 Da.

[47] The method of any one of embodiments 1-46, wherein the polyfunctional aziridine compound AZ comprising units (A) comprises one or more linking chains wherein each one of these linking chains links two of the structural units (A).

[48] The method of embodiments 47, wherein the linking chains consist of from 2 to 300 atoms, preferably from 5 to 250, or from 6 to 100 atoms; and wherein the atoms of the linking chains are C, N, O, S and/or P, preferably C, N and/or O.

[49] The method of any one of embodiments 47-48, wherein the number of consecutive atoms between the N atom of the urethane group in a structural unit (A) and the next N atom that is either present in the linking chain or is the N atom of the urethane group of another structural unit A is at most 9.

[50] The method of any one of embodiments 1-49, wherein the polyfunctional aziridine compound AZ comprises one or more connecting groups, wherein each one of these connecting groups connects two of the structural units (A), whereby a connecting group is defined as an array of consecutive functionalities connecting two structural units (A), and wherein the connecting groups consist of one or more functionaliies selected from the group consisting of aliphatic hydrocarbon functionality (preferably containing from 1 to 8 carbon atoms), cycloaliphatic hydrocarbon functionality (preferably containing from 4 to 10 carbon atoms), aromatic hydrocarbon functionality (preferably containing from 6 to 12 carbon atoms), isocyanurate functionality, iminooxadiazindione functionality, ether functionality, ester functionality, amide functionality, carbonate functionality, urethane functionality, urea functionality, biuret functionality, allophanate functionality, uretdione functionality and any combination thereof.

[51] The method of embodiment 50, wherein the connecting group consists of a consecutive array of aliphatic hydrocarbon functionality 1 (a linear C ₆ HI ), isocyanurate 2 (a cyclic C3N3O3) functionality and aliphatic hydrocarbon functionality 3 (a linear C6H12); or, wherein the connecting group consists of a consecutive array of aliphatic hydrocarbon functionality 1 (a branched C ₃ H ₆ ), aromatic hydrocarbon functionality 2 (a benzene ring) and aliphatic hydrocarbon functionality 3 (a branched C ₃ H ₆ ).

[52] The method of embodiment 49, wherein the connecting group consists of a consecutive array of aliphatic hydrocarbon functionality 1 (a linear C ₆ HI ₂ ), uretdione 2 (a cyclic C ₂ N ₂ 0 ₂ ) and aliphatic hydrocarbon functionality 3 (a linear C ₆ HI ₂ ).

[53] The method of any one of embodiments 49-52, wherein the polyfunctional aziridine compound has two structural units (A) and one connecting group connecting these two structural units, or the polyfunctional aziridine compound has three structural units (A) and three connecting groups, wherein each one of the three connecting groups connects a structural unit (A) with another structural unit (A).

[54] The method of any one of embodiments 49-53, wherein the connecting groups consist of at least one functionality selected from the group consisting of aliphatic hydrocarbon functionality (preferably containing from 1 to 8 carbon atoms), cycloaliphatic hydrocarbon functionality (preferably containing from 4 to 10 carbon atoms), aromatic hydrocarbon functionality (preferably containing from 6 to 12 carbon atoms), isocyanurate functionality, iminooxadiazindione functionality, urethane functionality, urea functionality, biuret functionality and any combination thereof.

[55] The method of any one of embodiments 49-54, wherein the connecting groups contain an isocyanurate functionality, an iminooxadiazindione functionality, a biuret functionality, an allophanate functionality or an uretdione functionality

[56] The method of any one of embodiments 49-55, wherein the connecting groups contain an isocyanurate functionality or an iminooxadiazindione functionality. [57] The method of any one of embodiments 49-56, wherein the polyfunctional aziridine compound has been obtained from the reaction product of one or more suitable compounds B and a hybrid isocyanurate such as a HDI/IPDI isocyanurate, containing a connecting group consisting of the array of a linear C ₆ HI (i.e. an aliphatic hydrocarbon functionality with 6 carbon atoms), an isocyanurate functionality (a cyclic C ₃ N ₃ O ₃ ) and a cycloaliphatic hydrocarbon functionality with 9 carbon atoms and an aliphatic hydrocarbon functionality with 1 carbon atom.

[58] The method of any one of embodiments 49-57, wherein the connecting groups have one or more substituents as pendant groups that enhance solubility of the compound in aqueous media, like an oligomer of ethylene oxide, vinyl pyrrolidone or an oxazoline, preferably the pendant groups contain ethylene oxide units.

[59] The method of any one of embodiments 49-58, wherein the connecting groups consist of at least one aliphatic hydrocarbon functionality and/or at least one cycloaliphatic hydrocarbon functionality, and optionally at least one aromatic hydrocarbon functionality and optionally an isocyanurate or iminooxadiazindione or allophanate or uretdione functionality.

[60] The method of any one of embodiments 49-59, wherein the connecting groups consist of at least one aliphatic hydrocarbon functionality and/or at least one cycloaliphatic hydrocarbon functionality, and optionally at least one aromatic hydrocarbon functionality and optionally an isocyanurate or iminooxadiazindione functionality.

[61] The method of any one of embodiments 49-60, wherein the connecting group consists of consecutive aliphatic hydrocarbon functionality, isocyanurate functionality, and aliphatic hydrocarbon functionality.

[62] The method of any one of embodiments 1-61, wherein the polyfunctional aziridine compound AZ has structural formula wherein Z is a molecular residue obtained by removing isocyanate reactive groups XH of a molecule; q is an integer from 2 to 6; i is the index for the different groups D and is an integer from 1 to q;

Di groups independently have the following structural formula wherein X is NRn, S or O, whereby Rn is H or an alkyl group with 1 to 4 carbon atoms; Y is an aromatic hydrocarbon group, an aliphatic hydrocarbon group, a cycloaliphatic hydrocarbon group or a combination thereof; j is an integer from 1 to p; p is an integer from 0 to 10; and m, R', R", Ri, R ₂ , R ₃ and R ₄ are as defined above.

[63] The method of embodiment 62, wherein the structural units D, are independently the same or different, preferably the structural units D, are the same.

[64] The method of embodiment 62 or 63, wherein the isocyanate reactive groups XH are hydroxy groups (X is O), primary amines (X is NH) or secondary amines (X is NRn wherein Rn is an alkyl group with 1 to 4 carbon atoms), preferably groups XH are hydroxy groups (X is O) and primary amines (X is NH).

[65] The method of any one of embodiments 62-64, wherein the molecule from which isocyanate reactive group are removed to obtain Z is a diol, a triol, a polyether with terminal isocyanate reactive groups, a polyamide with terminal isocyanate reactive groups, a polycarbonate with terminal isocyanate reactive groups, or a polysiloxane with terminal isocyanate reactive groups that are linked to the siloxane via at least one carbon atom.

[66] The method of any one of embodiments 62-65, wherein q is 2 or 3 and preferably q is 1.

[67] The method of any one of embodiments 62-66, wherein p is an integer from 0 to 10, preferably from 0 to 5, or from 0 to 3.

[68] The method of any one of embodiments 62-67, wherein p is 0 for all D,, and preferably m is 1.

[69] The method of any one of embodiments 62-68, wherein the total amount of cyclic structures (apart from the aziridine groups) present in the polyfunctional aziridine compound AZ is at most 3, preferably from 0 to 2, 1 or 2, or 1.

[70] The method of any one of embodiments 62-69, wherein the cyclic structures (apart from the aziridine groups) present in the polyfunctional aziridine compound AZ are an isocyanurate or an iminooxadiazindione.

[71] The method of any one of embodiments 1-70, wherein the polyfunctional aziridine compound AZ contains at least 5 mass%, preferably at least 5.5, 6, 9 or at least 12 mass%, and less than 25 or less than 20 mass % of urethane bonds.

[72] The method of any one of embodiments 1-71, wherein the polyfunctional aziridine compound AZ has an aziridine equivalent mass (molar mass of the polyfunctional aziridine compound divided by number of aziridinyl groups present in the polyfunctional aziridine compound) of at least 200 Da, preferably at least 230 or 260 Da, and at most 2500 Da, preferably at most 1000 or 500 Da.

[73] The method of any one of embodiments 1-72, wherein the polyfunctional aziridine compound AZ has been obtained by reacting at least a polyisocyanate and a compound B with the following structural formula: wherein the molar ratio of compound B to polyisocyanate is from 2 to 6, preferably from 2 to 4 or from 2 to 3, and wherein m, R', R", Ri, R ₂ , R ₃ and R ₄ are as defined above.

[74] The method of any one of embodiments 1-73, wherein the polyfunctional aziridine compound AZ contains less than 35 mass% of alkoxy poly(ethyleneglycol), preferably methoxy poly(ethyleneglycol) (MPEG), and/or poly(ethyleneglycol) (PEG) chains with a number average molar mass M _n higher than 2200 Da orhigherthan 1600 Da, preferably said amount is less than 15 mass% and more than 1 mass%, or more than 2, 3, 4, or 5 mass.

[75] The method of any one of embodiments 1-74, wherein the polyfunctional aziridine compound AZ contains methoxy poly(ethyleneglycol) (MPEG) and/or poly(ethyleneglycol)

(PEG) chains with a M _n lower than 1600 Da, preferably lower than 100, 770 or 570 Da.

[76] The method of any one of embodiments 1-75, wherein the polyfunctional aziridine compound AZ is represented by the structural formula: wherein the compound comprises at least one ethylene oxide oligomer as R”, preferably 2, 3 or an average of from 2 to 3 ethylene oxide oligomers as R”.

[77] The method of embodiment 76, wherein each ethylene oxide oligomer independently contains at most 35, 30, 25, 20 or 15 ethylene oxide units and at least 2, 3, 4 or 5 ethylene oxide units; or has a molar mass at least 85, 125, 170, 210 Da and at most 1500, 1300, 1100, 900 or 700 Da, like about 600 Da.

[78] The method of any one of embodiments 1-77, wherein the average number of aziridinyl groups in the polyfunctional aziridine cross-linker AZ is at least 1.8, preferably at least 2 or 2.2, and preferably less than 10, more preferably less than 6 or 4, or the average number of aziridinyl groups is from 2.2 to 3. [79] The method of any one of embodiments 1-78, wherein the calculated average amount of urethane bonds is at least 5 mass% (relative to the total mass of the polyfunctional aziridine compound AZ), preferably at least 5.5. 6, 9, or 12 mass% and less than 25 or 20 mass%.

[80] The method of any one of embodiments 1-79, wherein the polyfunctional aziridine compound AZ has a Brookfield viscosity of at least 500 mPa.s at 25 °C, preferably at least 1200 or 3000 mPa.s at 25 °C and at most 1000000, 100000, 30000, 10000 or at most 5000 mPa.s at 25 °C (as determined according to ISO 2555-89).

[81] The method of any one of embodiments 1-79, wherein the polyfunctional aziridine compound AZ has a Brookfield viscosity in the range of from 300 to 20000 mPas, preferably from 500 to 12000 or 700 to 3000 mPas (as measured with a Brookfield with spindle S63, at 25 °C at 80% solids, 20% in dimethyl formamide).

[82] The method of any one of claims 2-81 , wherein making the biopolymer composition comprises dissolving and/or dispersing the biopolymer material in an aqueous medium under such conditions, of for example temperature, time, pH, auxiliary equipment like a stirrer, and optionally additional components like a dispersion agent or a co-solvent, that a specific structural conformation of the biopolymer can be preserved.

[83] The method of any one of claims 2-82, wherein mixing a polyfunctional aziridine compound AZ into a solution or dispersion of biopolymer material comprises adding the compound as a pure material in liquid or solid form, or as a dispersion or solution, like an aqueous dispersion or solution.

[84] The method of any one of claims 2-83, wherein the solution or dispersion comprising biopolymer (material) and the aziridine compound or aziridine compound solution or dispersion are stored separately, and mixing of the polyfunctional aziridine compound into the solution or dispersion of biopolymer material is done just prior to a subsequent step of forming a coating or an article.

[85] The method of any one of claims 2-84, wherein a ratio of aziridinyl groups of the aziridine compound to functional groups of the biopolymer is from 0.01 to 5.0, preferably at least 0.05, 0.1 , 0.2, 0.25, or 0.3 and at most 4, 3, 2, 1.5, 0.95, 0.9, 0.85 or 0.8.

[86] The method of any one of claims 2-85, wherein a step of applying the mixture comprising biopolymer material and aziridine compound to a surface of a substrate is done using known application methods, like casting, brushing, dipping or spraying.

[87] The method of any one of claims 2-86, wherein the mixture comprising biopolymer material and aziridine compound is applied to a specific part of a surface of a substrate, or to all surface of a substrate. [88] The method of any one of claims 2-87, wherein the substrate is a medical device or implant, which device or a part thereof can have been made from natural or synthetic materials, including metals such as titanium and stainless-steel grades, like surgical steel; ceramics such as calcium phosphates, like hydroxyapatite, TCP, bioglass, and mixtures thereof; and organic polymers such as bioinert polymers like PEEK, UHMWPE, and biodegradable polymers like copolymers comprising lactic acid, polyhydroxyesters, and polyesteramide copolymers including copolymers comprising natural amino acids.

[89] The method of any one of claims 2-88, wherein the biopolymer composition is a solution or dispersion comprising the biopolymer material and volatiles like water are removed therefrom, for example by a freeze-drying, to result in a solid, porous article or coating.

[90] An article or a component comprising cross-linked biopolymer as obtainable by or as obtained with the methods according to any one of embodiments 1-89, such as a porous collagen-based sheet or other structure for use in for example tissue engineering, or (a component for) an orthopedic or dental implant having a collagen-based coating on at least part of its surface.

[91] A medical device or medical implant, comprising an article or a component comprising cross-linked biopolymer as obtainable by or as obtained with the methods according to any one of embodiments 1-89.

[92] The medical device or medical implant according to embodiment 91 , comprising on at least part of its surface a coating comprising cross-linked biopolymer.

[93] The medical device or implant according to embodiment 92, wherein the coating layer has a thickness of from 0.1 to 200 pm, preferably coating thickness is at least 0.5, 1 , 2, 5 or 10 pm and at most 150, 100, 50 or 25 pm.

[94] The medical device or implant according to any one of embodiments 91-93, which has at least partly been made from one or more different materials, including metals, ceramics and organic polymers, and which may have various physical shapes, including non-porous, monolithic implants like a titanium screw, and porous structures like fibrous constructs such as sutures or fabrics comprising polyester or polyethylene yarns.

The present invention is now further illustrated by reference to the following examples and comparative experiments, which should not be construed as in any way limiting the scope of the claims. Unless otherwise specified, all parts, percentages and ratios are on a mass basis and relative to the total composition. Experimental part

Components and abbreviations used

Acid-soluble collagen, derived from bovine hides, obtainable as Semed S from DSM Biomedical Inc. (Exton, PA (USA)), was used as collagen.

A mixture of mono-, di- and tri-functional aziridine compounds (AZ-1) having below presented structures was prepared as described in W02020/020714A1.

Analogously, a mixture of mono-, di- and tri-functional aziridine compounds (AZ-1b) of below shown structures was prepared, which contains an average of 11 ethylene oxide units (vs 8 units for AZ-1).

These pendant oligo(ethylene oxide) chains increase hydrophilic character of the compounds and enable mixing the compounds with an aqueous collagen dispersion without further additives.

The below indicated trifunctional aziridine compound (AZ-2), was also prepared as described in W02020/020714A1 :

An aqueous dispersion of AZ-2 was made using a nonionic polyalkylene oxide blockcopolymer (Maxemul™ 7101 ; Croda Coatings & Polymers) as dispersing agent.

Toxicity testing

Genotoxicity of compounds has been measured by the ToxTracker® assay (Toxys, Leiden, the Netherlands); and results are summarized in Table 1. Herein Bscl2 and Rtkn refer to specific genes linked to reporter genes for the detection of DNA damage (biomarkers); and genotoxicity was evaluated using concentrations that were pre-determined to induce about 10, 20 and 50% cytotoxicity; as further described in the experimental part of W02020/020714A1. The results presented in Table 1 , show a negative induction level of the biomarkers at all compositions of the 3 compounds AZ-1 , AZ-1 b and AZ-2 evaluated; that is less than 1.5-fold induction. Compounds AZ are thus concluded to be not genotoxic. As a comparative example, the known aziridine compound CX-100 was found to show > 1 .5-fold induction for multiple tests as reported in W02020/020714A1 . Table 1

For corroborative purposes, mutagenicity of the crosslinker compounds was also assessed; using the established Ames test (Bacterial Reversion Assay) according to the following guidelines: OECD Guideline 471 . Genetic Toxicology: Bacterial Reverse Mutation Test. (Adopted

July 21 , 1997); and

EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008. These tests showed that the crosslinkers AZ-1 , AZ-1 b and AZ-2, and other compounds

AZ as described herein above are not mutagenic.

Examples and comparative experiments

Comparative experiment 1 A 1 mass% aqueous dispersion of collagen was made by mixing acid-soluble collagen with MilliQ water set to pH 2.4 with HCI during 1 h at room temperature on a rollerbank, followed by standing overnight in a refrigerator to further dissolve, after which the pH was brought to 3.4 by adding NaOH. Adhesion of a coating to a metal surface was evaluated by pipetting 1.6 ml of the collagen dispersion on 10 x 10 cm cleaned stainless steel plates (Goodfellow, LS314334 L O, AISI 316 (Fe/Crie/Niio/Mo ₃ ); thickness 0.25 mm; cleaned by sonication for 10 minutes in 500 ml of following subsequently IPA, MNNQ/HNO3 (78/22 v/v), demineralized water, MilliQ water, and IPA/MilliQ (70/30 v/v); followed by drying for 10 minutes at 80 °C), spreading the dispersion into a coating area of about 1.5 x 8 cm , and drying at room temperature during about 64 h and during 2 h at room temperature and under vacuum.

Example 2

Collagen dispersion and coatings on stainless steel plates were made analogously to Comparative experiment 1 , but 1 equivalent of crosslinker AZ-1 was added to the collagen dispersion (that is 1 mole of reactive groups in the aziridine compound per 1 mole of carboxylic acid groups originating from aspartic acid and glutamic acid in the collagen). The mixture was vigorously stirred and then spun down in an Eppendorf centrifuge at 2000 rpm for 2 min. to remove air bubbles before applying to the steel plate.

Example 3

Collagen dispersion and coatings on stainless steel plates were made analogously to Comparative experiment 1 , but 1 equivalent of crosslinker AZ-2 was added to the collagen dispersion (that is 1 mole of reactive groups in the aziridine compound per 1 mole of carboxylic acid groups originating from aspartic acid and glutamic acid in the collagen). The mixture was vigorously stirred and then spun down in an Eppendorf centrifuge at 2000 rpm for 2 min. to remove air bubbles before applying to the steel plate.

Comparative experiment 4

Collagen dispersion and coatings on stainless steel plates were made analogously to Comparative experiment 1 , but 1 equivalent of glutaraldehyde crosslinker was added to the collagen dispersion (that is 1 mole of reactive groups per 1 mole of carboxylic acid groups originating from aspartic acid and glutamic acid in the collagen). The mixture was vigorously stirred and then spun down in an Eppendorf centrifuge at 2000 rpm for 2 min. to remove air bubbles before applying to the steel plate.

The coated plates were subsequently tested on adhesion as indicated below. Results of visually evaluating the stained plates are summarized in Table 1.

Test 1 : only staining of the coatings to check homogeneity.

Test 2: dry tape test; by applying a piece of Scotch tape to the coating, rubbing firmly except for the initial < 1cm; peeling off the tape by hand in one smooth motion. Test 3: dissolution in PBS; by incubating the applied collagen in PBS at 37 °C during 16 h on a shaker; rinsing with demi-water; air drying during 2 h.

Test 4: tape test; by incubating the applied collagen in PBS at 37 °C during 16 h on a shaker; rinsing with demi-water; air drying during 2 h; and performing a tape test as in test 2. Test 5: wet scraping; by incubating the applied collagen in PBS at 37 °C during 16 h on a shaker; rinsing with demi-water; scraping the film by hand with a spatulum having a 1 cm wide straight end; and rubbing non-scraped parts of the coating with a finger covered by a nitrile glove.

In all tests the coatings were subsequently stained by submersing in a 0.3 mass% Rose Bengal solution in methanol during 10 min., washing twice with methanol; and air drying at room temperature. All samples were photographed and visually evaluated and rated. Results as summarized in Table 2 indicate that the collagen coatings that have been cross-linked with aziridine compound show superior adhesion to stainless steel compared to non-crosslinked coating and glutaraldehyde cross-linked coating, in both dry and wet state. Figure 1 illustrates these results, by showing test plates each having a coating of Ce1 ,

Examples 2 and 3, and Ce 4 (from left to right) after tests 1-5.

Table 2