Functionalized recombinant bacterial collagen-like proteins

Abstract

A recombinant bacterial collagen-like protein, preferably having an amino acid sequence at least > = 60% identical to the amino acid sequence of SEQ ID NO: 1, where the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1, where the recombinant collagen-like protein is functionalized with at least one non-terminal alkenyl group. The present invention relates to a functionalized recombinant bacterial collagen-like protein, a bio-ink comprising the functionalized recombinant bacterial collagen-like protein, at least one solvent and at least one photoinitiator, a method for producing a hydrogel by crosslinking the functionalized recombinant bacterial protein or the bio-ink and the hydrogel obtained therefrom. In addition, the scaffold for tissue engineering comprises a hydrogel.

Description

Invention Field

[0001] This invention relates to bacterial functionalized recombinant collagen-like proteins (CLPs) as cell-encapsulating bioinks, and their applications in the preparation of 3D scaffolds, in vitro models, and tissue grafts. Background of the Invention

[0003] Bio-inks typically used for bioprinting are mainly synthetic thermoplastic polymers or animal-derived proteins, such as collagen, gelatin, (meth)acryloyl-functionalized animal-derived collagen, or gelatin methacrylate.

[0004] Collagen-based hydrogels are the optimal scaffold materials for cell encapsulation. Their biodegradability and biocompatibility allow for optimal material conditions for bioprinting, while multiple cell interaction domains (naturally present in collagen) enable cell diffusion and scaffold colonization.

[0005] US2016 / 0051727 discloses a collagen-based polymer material comprising collagen molecules and / or collagen-derived molecules, said collagen molecules and / or collagen-derived molecules being functionalized by adding one or more olefinic unsaturated groups and crosslinked via said groups.

[0006] US 2020 / 0179562 discloses curable recombinant human collagen functionalized with methacrylamide, which can be formulated into inks for additive manufacturing and printed into three-dimensional objects.

[0007] US 2016 / 0193384 discloses the production of hydrogels by photocrosslinking methacryloyl or acryloyl collagen and synthetic polymers. Such hydrogels can be used as 3D scaffolds and implants, and they are suitable as inks for 3D printing to fabricate complex 3D structures or for incorporating cells into structures.

[0008] Photocurable gelatin-based hydrogels have been identified as powerful bioinks in tissue engineering due to their excellent biocompatibility, biodegradability, photoresponsiveness, thermosensitivity, and bioprinting properties. While gelatin methacryloyl (GelMA) has been the gold standard for many years, thiol-ene hydrogel systems based on norbornene-functionalized gelatin (GelNB) and thiol crosslinking agents have recently gained increasing importance. (e.g., 2021). The researchers disclosed a highly reproducible water-based synthesis of GelNB, covering a wide range of functionalization levels (DoF: 20% to 97%) and its mixing with thiolized gelatin (GelS), resulting in ultrafast curing photoclick hydrogels GelNB / GelS.

[0009] However, the collagen used in the aforementioned prior art is derived from human or animal sources. Such human or animal-derived collagens are not well-defined due to their natural origin, are viscous, and have poor water solubility under physiological conditions. Because of their viscosity, significant forces are required during the printing process to extrude or jet these collagen mixtures. If cells are co-printed in the same mixture, this results in greater shear stress and thus reduced cell viability. The low pH required to dissolve animal-derived collagen also makes increasing concentrations challenging. Another disadvantage due to the origin is the risk of quality variation and disease transmission.

[0010] Bacterial collagen-like proteins (CLPs) possess interesting mechanical properties, similar to those of collagen in higher eukaryotes, without requiring the complex maturation steps necessary for their eukaryotic counterparts. CLPs exhibit a common structure: two α-helices, stabilized together, forming a "V-domain," followed by a rod-shaped collagen domain (CL). Following the collagen domain, membrane anchors (e.g., GPI-like structures) are typically present at the C-terminus of the protein.

[0011] The most industrially relevant CLP is Scl2 from Streptococcus pyogenes. As described in various publications (Lukomski et al. 2002, Brodsky et al. 2009), the current understanding is that a V domain is required to fold the three Scl2 protein monomers into a triple helix structure in vitro.

[0012] While bioinks based on human or animal-derived collagen are widely used, the demand for GMP-compliant bioinks remains unmet to facilitate the translation of bioprinted clinical products from benchmark to bedside, particularly water-soluble biomaterials that can form hydrogels and thus support cell encapsulation and bioprinting. Desired properties include consistent batch quality, high purity, and good solubility under conditions suitable for encapsulating cells.

[0013] Therefore, one object of the present invention is to provide a protein that can be formulated into a bio-ink and cross-linked into a hydrogel, and can at least reduce the disadvantages of prior art materials, particularly animal-derived collagen. Invention Overview

[0015] Therefore, in one aspect, the present invention relates to recombinant bacterial collagen-like protein, which preferably comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1, wherein the recombinant collagen-like protein is functionalized with at least one non-terminal alkenyl group.

[0016] In one embodiment of the invention, the amino acid sequence is at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0017] In another embodiment, the degree of functionalization of the at least one non-terminal olefinic group ranges from 5% to 100% of the sum of primary amines comprising the N-terminal primary amine group and the primary amine group of lysine residues of the entire recombinant collagen-like protein.

[0018] In one embodiment of the invention, the at least one non-terminal olefin is selected from linear, branched or cyclic non-terminal olefins, preferably from norbornene or its derivatives, or combinations thereof.

[0019] In another aspect, the present invention relates to a bio-ink composition comprising:

[0020] a) about 0.25 to about 20% by weight of at least one functionalized recombinant bacterial collagen-like protein according to the invention;

[0021] b) Approximately 75% to approximately 99% by weight of an aqueous solvent;

[0022] c) at least one photoinitiator, in amounts of about 0.01 to about 2% by weight;

[0023] d) about 0% to about 5% by weight of at least one photocrosslinking polymer or other photocrosslinking peptide;

[0024] e) about 0 to about 15% by weight of at least one compound selected from additives, rheology modifiers, biopolymers, gelling enhancers, bioactive components, peptides, nanocellulose and / or cells.

[0025] The condition is that the total amount of all components of the bio-ink is 100% by weight.

[0026] In one embodiment of the invention, the bio-ink composition further comprises about 0.125 to about 10% by weight of at least one additional recombinant bacterial collagen-like protein, wherein the additional recombinant bacterial collagen-like protein comprises a functionalization different from that of the functionalized recombinant bacterial collagen-like protein of a).

[0027] In another embodiment of the invention, the at least one additional functionalized recombinant bacterial collagen-like protein is a recombinant bacterial collagen-like protein comprising at least 60% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1, and wherein the recombinant collagen-like protein is functionalized with at least one thiol group.

[0028] In one embodiment of the invention, the at least one additional functionalized recombinant bacterial collagen-like protein is a recombinant bacterial collagen-like protein comprising at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% of the same amino acid sequence as SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0029] In one embodiment of the bio-ink composition of the present invention, the additional functionalized recombinant bacterial collagen-like protein is functionalized to a degree of 5% to 100% of the sum of primary amines of the recombinant collagen-like protein containing primary amine groups comprising N-terminal primary amine and lysine residues.

[0030] In one embodiment, the bio-ink composition further comprises thiol molecules, thiol dimers, or thiol polymers, preferably multi-arm PEG, such as 4-ArmPEG-SH or dithiothreitol (DTT).

[0031] In another aspect, the present invention relates to a method for producing hydrogels by crosslinking, preferably photocrosslinking, the functionalized recombinant bacterial collagen-like proteins or bioink compositions of the present invention.

[0032] In one embodiment of the method of the present invention, the bio-ink composition is crosslinked using ultraviolet or visible light, preferably blue light.

[0033] In another respect, the present invention relates to hydrogels obtained by the method according to the invention, preferably injectable hydrogels.

[0034] In one embodiment of the hydrogel of the present invention, the hydrogel further comprises nanocellulose, peptides, or mixtures thereof.

[0035] In another aspect, the present invention relates to a scaffold for tissue engineering comprising a hydrogel according to the invention.

[0036] Attached Figure Description

[0037] Figure 1 The reaction scheme of the present invention is to modify the recombinant bacterial collagen-like protein of the present invention with norbornene groups to obtain the recombinant bacterial collagen-like protein (rColN) containing norbornene groups of the present invention.

[0038] Figure 2 Compared to unmodified recombinant bacterial collagen-like protein (CLP), the functionalized recombinant bacterial collagen-like protein (rColN) of the present invention containing norbornene groups has the advantages of the present invention. 1 H-NMR example.

[0039] Figure 33T3 cell viability 24 hours after exposure to different concentrations of unmodified CLP and rColN. The applied molar ratios were equal to the following DoFs, in %: 1:0.1 = 15%; 1:0.25 = 31%; 1:0.5 = 52%; 1:0.75 = 76% and 1:1 = 91%.

[0040] Figure 4 The reaction schemes for the photo-induced crosslinking reaction of functionalized recombinant bacterial collagen-like protein (rColS) containing thiol groups and other recombinant bacterial collagen-like protein (rColN) containing norbornene groups of the present invention with photoinitiator LAP (phenyl-2,4,6-trimethylbenzoyl lithium phosphine).

[0041] Figure 5 Human dermal fibroblasts (HFF cells) stained after culturing in rColN hydrogel for 7 and 14 days showed the formation of a cell network. Nearly 100% of all cells were identified as viable.

[0042] Figure 6 (A) Print the rColN mixture described herein using different techniques. (B) Print the rColN mixture (1% (w / v) rColN (48%), 1% (w / v) rColS (45%) and 0.03% LAP in 1×PBS by drop-on printing as needed.

[0043] Figure 7 : Reaction protocol for hydrogel synthesis using rColN and 4-arm-PEG-SH, 10kDa (JenKemTechnology USA)).

[0044] Figure 8 The correlation between different CLP and rColN concentrations dissolved in 1xPBS and the viscosity measured using an Anton Paar MCR 502WESP system with a 50 mm plate-cone system (1° angle; N=3). rColN_high equals 77% DoF, while rColN_low equals 21% DoF.

[0045] Figure 9 Example measurement of shear storage modulus using ElastoSens Bio online measurement.

[0046] sequence

[0047] SEQ ID NO: 1 Streptococcus pyogenes collagen-like protein (CLP), full-length protein

[0048] SEQ ID NO: 2 Streptococcus pyogenes CLP, truncated 3

[0049] SEQ ID NO: 3 Streptococcus pyogenes CLP, truncated 5

[0050] SEQ ID NO: 4 Streptococcus pyogenes CLP, without V domain Invention Details

[0052] Collagen-based hydrogels are optimal scaffold materials for cell encapsulation. Their biodegradability and biocompatibility allow for optimal material conditions for bioprinting, while multiple cell-cell interaction domains (naturally present in collagen) enable cell diffusion and scaffold colonization. Currently available and described collagen-based hydrogels are based on human or animal sources. These human or animal-derived collagens have various drawbacks, such as varying quality, high viscosity, poor water solubility under physiological conditions, and the potential to carry diseases. Due to their viscosity, significant forces are required during the printing process to extrude or jet the viscous collagen mixture. If cells are co-printed in the same mixture, this results in greater shear stress (and thus reduced cell viability). Furthermore, the low pH required to dissolve animal-derived collagen makes increasing concentrations challenging.

[0053] Due to the great need for optimized bio-inks that overcome the aforementioned drawbacks, the inventors have proposed providing a recombinant collagen-like protein that is bacterially derived, thus serving as a vegetarian alternative to existing animal-derived collagen raw materials, and which can be functionalized and used to prepare optimized bio-inks for cell encapsulation and hydrogel production.

[0054] Recombinant collagen-like protein (CLP)

[0055] The most industrially relevant CLP for *Streptococcus pyogenes* Scl2 contains a large V domain, which constitutes approximately one-third of the entire Scl2 sequence and hinders protein transport from the *Pichia pastoris* host. This necessitates a complex downstream process involving cell lysis to remove the target protein from the cell. Furthermore, the V domain itself is pathogenic and therefore needs to be removed during purification via protease digestion. The use of proteases is very expensive, and they must be removed during downstream processing. Therefore, producing purified Scl2 from the full-length protein including the V domain requires additional costly downstream process steps.

[0056] The inventors have now surprisingly discovered that truncated variants of collagen-like proteins, including variants with or without any V domain, lead to increased production and secretion of collagen-like proteins into the fermentation medium. Even more surprisingly, the truncated variants fold correctly even in the absence of the V domain.

[0057] In addition to what has been previously described in the prior art, the V domain therefore appears not to require the three Scl2 protein monomers to be properly folded into a triple helix structure in vitro, which opens up the possibility of overcoming the various challenges arising from first expressing the full-length Scl2 protein and then removing the V domain.

[0058] Therefore, in one aspect, the present invention relates to recombinant bacterial collagen-like protein, which preferably comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1, wherein the recombinant collagen-like protein is functionalized with at least one non-terminal alkenyl group.

[0059] In one aspect, it is preferred that the recombinant bacterial collagen-like protein contains a deletion of 38 to 90 amino acids at the N-terminus of the amino acid sequence shown in SEQ ID NO:1, where SEQ ID NO:1 depicts the amino acid sequence of the full-length CLP. Preferably, the deletion is 38-74 amino acids. This includes a complete deletion of the N-terminal V-domain (containing 74 amino acids) and varying truncations of the V-domain of at least 38 amino acids.

[0060] The amino acid sequence of the recombinant bacterial collagen-like protein of the present invention is preferably at least 60% identical to the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. SEQ ID NO: 2 and SEQ ID NO: 3 are truncated forms of Scl2, wherein the following amino acids have been deleted:

[0061] SEQ ID NO: 2 is based on SEQ ID NO: 1, with amino acids (38 aa) deleted at positions 13-50.

[0062] SEQ ID NO: 3 is based on SEQ ID NO: 1, with amino acids (75aa) deleted at positions 1-74.

[0063] SEQ ID NO: 4 is based on SEQ ID NO. 1, with aa 1-90 (90aa) missing.

[0064] It should be understood that the SEQ ID NO shown herein describes the amino acid sequence prior to functionalization with at least one non-terminal olefinic group, rather than the functionalized recombinant bacterial collagen-like protein.

[0065] In a preferred embodiment, the recombinant bacterial collagen-like protein comprises at least 90%, 91%, 92%, 93%, 94%, 95%, or 96% of the amino acid sequence identical to that of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Preferably, the recombinant bacterial collagen-like protein comprises at least 97%, more preferably at least 98%, and most preferably at least 99% of the amino acid sequence identical to that of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0066] In the most preferred embodiment, the recombinant bacterial collagen-like protein consists of an amino acid sequence according to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0067] In another preferred embodiment, other truncated variants of collagen-like proteins from Streptococcus pyogenes are used as the basis for preparing recombinant bacterial collagen-like proteins functionalized with at least one non-terminal olefinic group, as further described above.

[0068] Therefore, the present invention also includes variants of recombinant bacterial collagen-like proteins according to SEQ ID NO: 1 to 4, wherein said variants preferably comprise one or more amino acid exchanges, insertions, and / or deletions. In a preferred embodiment, such variants contain up to 5, up to 4, up to 3, or up to 2 amino acid exchanges, insertions, and / or deletions.

[0069] In another preferred embodiment of the invention, the variant of the recombinant bacterial collagen-like protein comprises at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, preferably at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, more preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and most preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the same amino acid sequence as SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0070] The recombinant bacterial collagen-like protein (CLP) of the present invention can be produced in a method comprising the following steps:

[0071] a) Fermentation in a culture medium of bacterial, yeast, or plant host cells expressing a CLP having at least ≥60% identical amino acid sequence to that of SEQ ID NO:1, wherein the amino acid sequence includes a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:1.

[0072] b) Accumulation of bacterial CLP in the culture medium, from which fermentation broth is obtained.

[0073] c) Isolate bacteria, yeast, or plant host cells from the fermentation broth.

[0074] d) Incubate the fermentation broth at no more than 25°C for at least 1 hour to fold the CLP.

[0075] e) Optionally purify bacterial CLP by at least one of the following: solvent precipitation, tangential flow filtration (TFF), ion exchange chromatography, or reversed-phase chromatography.

[0076] In a preferred embodiment, the folding of the CLP in step d) is performed at a temperature of -80°C to 25°C, preferably 0°C to 20°C. In a preferred configuration, the folding is performed in the presence of glycerol or a salt.

[0077] In another preferred embodiment, the folding of CLP in step d) is carried out for 1 hour to 48 hours, preferably 1 hour to 24 hours.

[0078] In another preferred embodiment, the folding of CLP in step d) is carried out at a CLP concentration of at least 1 mg / ml, preferably at least 4 mg / ml.

[0079] In a preferred embodiment, the host cell is a microorganism of the species P. pastoris, E. coli, P. putida, or C. glutamicum, containing any of the polypeptides described in this invention.

[0080] In a preferred embodiment, the microorganism is a yeast or bacterial cell of the genus Pichia pastoris, preferably Escherichia coli, Corynebacterium, or Brachybacterium.

[0081] Microorganisms can be microorganisms in which the nucleotide sequence encoding CLP is overexpressed.

[0082] Functionalization

[0083] The recombinant bacterial collagen-like protein of the present invention and as described above is functionalized with at least one non-terminal alkenyl group. The non-terminal alkene may be selected from straight-chain, branched, or cyclic non-terminal alkenes, preferably cyclic alkenes having 4 to 16 or 5 to 12 carbon atoms, more preferably norbornene or its derivatives, or combinations thereof.

[0084] In a preferred embodiment of the recombinant bacterial collagen-like protein of the present invention, the non-terminal olefinic group is a norbornene group. The term "norbornene," also known as norbornene or norbornene, refers to a cyclohexene ring with a bridging methylene group at the para position.

[0085] Compared to conventional free-radical chain polymerization of known olefin-containing biomaterials such as animal-derived GelMA or ColMA (methacrylated gelatin and collagen), the non-terminal olefins, particularly norbornene, conjugation produces bacterial collagen-like proteins that maintain their high solubility even at higher concentrations. This results in excellent materials for use in 3D printing techniques described further below. In summary, due to their low viscosity, the functionalized recombinant bacterial collagen-like proteins of this invention provide unique collagen-based materials for 3D printing techniques designed to handle low-viscosity solutions, such as stereolithography (SLA), digital light processing (DLP), and droplet-based bioprinting (jetting or on-demand ink delivery). These techniques are highly compatible with novel synthetic rColN materials.

[0086] The functionalized recombinant bacterial collagen-like protein according to the invention can be prepared by reacting the recombinant bacterial collagen-like protein as described above with at least one non-terminal olefin, preferably norbornene. Molecules particularly suitable for modification with norbornene are 5-norbornene-2-NHS ester, 5-norbornene-2,3-dicarboxylic anhydride, and 5-norbornene-2-carboxylic acid.

[0087] In a preferred embodiment, the functionalized recombinant bacterial collagen-like protein according to the invention can be prepared by reacting the recombinant bacterial collagen-like protein as described above with 5-norbornene-2-NHS ester (Nor-NHS). EDC-HCl (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) and NHS can be used to activate norbornene acid to obtain Nor-NHS. In such a preferred embodiment, only the norbornene unit is introduced into the recombinant bacterial collagen-like protein without any additional carboxylic acid. This results in a smaller change in the isoelectric point compared to the reaction product synthesized with 5-norbornene-2,3-dicarboxylic anhydride (which would introduce additional acidic side chains). The pH is maintained at 7 to 8, which is less harmful to the protein. Undesirable side reactions may occur as the pH increases. The reaction is kept at a constant RT (25°C) and no additional cooling to 4°C is required. Furthermore, the starting materials do not need to be dissolved in acetic acid, which has high viscosity, but are allowed to dissolve in water at a neutral pH. The same applies to the reaction product. This is particularly advantageous when encapsulating cells.

[0088] like Figure 1 The schematic depiction shows non-terminal olefinic groups, particularly norbornene functional groups, conjugated with primary amine groups present in lysine residues and the N-terminus of proteins.

[0089] The degree of functionalization of recombinant bacterial collagen-like proteins can be controlled by adjusting the molar ratio of primary amines present in the recombinant bacterial collagen-like proteins to added norbornene units, such as Nor-NHS. After the functionalization step, the mixture preferably undergoes percolation or dialysis to remove byproducts. The degree of functionalization can be determined by known methods, such as trinitrobenzenesulfonic acid determination, NMR, or HPLC-MS.

[0090] Due to the high solubility of recombinant bacterial collagen-like proteins in neutral and alkaline buffers, an unexpectedly high degree of functionalization up to 100% can be achieved, while minimizing damage to the triple-helix protein structure.

[0091] In another embodiment, the functionalization degree of the at least one non-terminal olefinic group is 5% to 100% of the sum of primary amines containing N-terminal primary amine groups and lysine residues in the entire recombinant collagen-like protein.

[0092] In a preferred embodiment of the invention, the degree of functionalization is at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% of the primary amine group on the N-terminus of the recombinant collagen-like protein.

[0093] In another preferred embodiment, the degree of functionalization is 10% to 90%, 20% to 80%, 25% to 75%, 30% to 70%, or 40% to 60%.

[0094] As further described in detail in Example 1, the degree of functionalization can be altered by changing the reaction conditions. Recombinant collagens with different degrees of functionalization (DoF) were obtained by varying the molar ratio of Nor-NHS to -NH2 groups in the recombinant collagen. For example, when the ratio of the amino groups of recombinant collagen to Nor-NHS was maintained at 1:0.25, a recombinant collagen with approximately 31% functionalization was synthesized. Increasing the ratio to approximately 1:0.75 resulted in 76% DoF.

[0095] In contrast, animal-derived collagen typically contains only about 10 to 20% functionalization because extreme reaction conditions denature the triple-helix protein structure, resulting in gelatin with lower mechanical rigidity. Therefore, the high degree of modification achievable with the functionalized bacterial collagen-like protein of this invention is a further advantage over animal-derived collagen.

[0096] Different degrees of functionalization (DoF) allow for tuning of the stiffness of the resulting hydrogels produced using the functionalized bacterial collagen-like proteins of this invention. Therefore, the materials of this invention offer significantly greater variability for a wide range of applications.

[0097] Another advantage of the recombinant bacterial collagen-like protein functionalized with non-terminal alkenyl groups, preferably norbornene groups, according to the invention is that it is soluble in water and aqueous buffer solutions at neutral or alkaline pH values, particularly at physiologically relevant pH values ​​of 7 to 7.4.

[0098] Bio-inks and hydrogels

[0099] Different cross-linking methods for proteins are known in the literature for synthesizing collagen hydrogels, but only a few are suitable for 3D bioprinting.

[0100] In another aspect, the present invention relates to a bio-ink composition comprising:

[0101] f) about 0.25 to about 20% by weight of at least one functionalized recombinant bacterial collagen-like protein according to the invention;

[0102] g) about 75 to about 99% by weight of aqueous solvent;

[0103] h) at least one photoinitiator, in amounts from about 0.01 to about 2% by weight;

[0104] i) about 0% to about 5% by weight of at least one photocrosslinking polymer or other photocrosslinking peptide;

[0105] j) about 0 to about 15% by weight of at least one compound selected from additives, rheology modifiers, biopolymers, gelling enhancers, bioactive components, peptides, nanocellulose and / or cells.

[0106] The condition is that the total weight of all components of the bio-ink is 100%.

[0107] The aqueous solvent can be water, any aqueous buffer system such as 1xPBS buffer or HEPES buffer, or it can be a culture medium. Buffer systems and culture media are well known in the prior art and can be selected according to the application required by the technician.

[0108] In a preferred embodiment, the aqueous solvent is water.

[0109] In one embodiment of the invention, the bio-ink composition further comprises about 0.125 to about 10% by weight of an additional recombinant bacterial collagen-like protein, wherein the additional recombinant bacterial collagen-like protein comprises a functionalization different from that of the functionalized recombinant bacterial collagen-like protein of a).

[0110] In another embodiment of the invention, the additional functionalized recombinant bacterial collagen-like protein is a recombinant bacterial collagen-like protein comprising at least 60% of the same amino acid sequence as the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1, and wherein the recombinant collagen-like protein is functionalized with at least one thiol group.

[0111] The term "thiol group" refers to any organosulfur compound of the form R-SH, where R represents an alkyl group or other organic substituent designed for reaction with primary amines. More preferably, recombinant bacterial collagen-like proteins are functionalized with a thiolactone group. The term "thiolactone group" refers to a lactone analog where an oxygen atom is replaced by a sulfur atom and the sulfur atom is adjacent to a carbonyl group within the ring system. Preferred thiolactones are homocysteine ​​thiolactones and their derivatives, with N-acetylhomocysteine ​​thiolactone being the most preferred.

[0112] In one embodiment of the bio-ink composition of the present invention, the additionally functionalized recombinant bacterial collagen-like protein is functionalized to a degree of 5% to 100% of the sum of primary amines containing the N-terminal primary amine and the primary amine group of lysine residues of the recombinant collagen-like protein.

[0113] In a preferred embodiment of the invention, the degree of functionalization is 5% to 90% of the sum of primary amines comprising the N-terminal primary amine group and the primary amine group of lysine residues of the entire recombinant collagen-like protein.

[0114] In a preferred embodiment of the invention, the degree of functionalization is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total primary amine groups of the recombinant collagen-like protein.

[0115] In a preferred embodiment, the degree of functionalization is 10% to 90%, 20% to 80%, 25% to 75%, 30% to 70%, or 40% to 60%.

[0116] In one embodiment, the bio-ink composition further comprises thiol molecules, thiol dimers, or thiol polymers, preferably multi-arm PEG, such as 4-ArmPEG-SH or dithiothreitol (DTT).

[0117] In one embodiment of the invention, the at least one additional functionalized recombinant bacterial collagen-like protein is a recombinant bacterial collagen-like protein comprising at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% of the same amino acid sequence as SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0118] In the bio-ink composition, non-terminated olefin (preferably norbornene) groups can form polymeric cross-linked networks with functionalized recombinant bacterial collagen-like protein molecules or with other components in the bio-ink composition in the presence of free radicals, anions, nucleophiles, or combinations thereof.

[0119] The bio-ink composition of the present invention contains a photoinitiator, preferably a free radical photoinitiator. The amount of photoinitiator added to the bio-ink composition is in the range of 0.01% to 2% by weight, based on the total weight of the composition. When irradiated with photochemical radiation, the photoinitiator is capable of generating free radicals.

[0120] It should be understood that adding at least one photocrosslinking polymer or other photocrosslinking peptide (component d) to the bioink composition of the present invention is optional, since the functionalized recombinant bacterial collagen-like protein according to the present invention can crosslink with itself.

[0121] Preferably, the photocrosslinking polymer of component d) is selected from natural (e.g., hyaluronic acid methacrylate) and synthetic (e.g., acrylate and methacrylate derivatives of poly(ethylene glycol)) polymers, and is mixed into the composition to impart additional biological properties (e.g., antifouling) and modulate physical properties (e.g., degradability and swelling behavior in water).

[0122] Synthetic peptides with photocrosslinking groups can be incorporated to impart additional biological activities, such as cell adhesion, stem cell differentiation, and enzymatic degradation. Biological activity is broadly described as cell adhesion and enzymatic degradation. Biological activity can be tailored by adding cell-interacting ligands during the photocrosslinking process.

[0123] Depending on the chosen photoinitiator, photopolymerization or photocrosslinking is preferably triggered by exposure to UV or visible light. A method for producing hydrogels by photocrosslinking the functionalized recombinant bacterial collagen-like proteins according to the invention also forms part of this invention. Similarly, a method for preparing hydrogels from the bio-ink compositions according to the invention by crosslinking (preferably photocrosslinking) also forms part of this invention.

[0124] In one embodiment of the method of the present invention, a UV photocrosslinking bio-ink composition is used.

[0125] In one embodiment of the method of the present invention, visible light, preferably blue light (380-500 nm), is used to crosslink the bio-ink composition. A wavelength of 390-410 nm is preferred, and 405 nm is most preferred.

[0126] Bioink compositions can be formulated by dissolving the functionalized recombinant bacterial collagen-like protein according to the invention in an aqueous solution and adding a water-soluble photoinitiator, such as lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP) or 1-[4-(2-methoxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959). Other suitable photoinitiators according to the invention are 2,2'-azobis[2-methyl-n-(2-hydroxyethyl)propionamide], tris(2,2-bipyridine)ruthenium(II) hexahydrate, eosin Y, Ivocerin, ZnTTP, and other Irgacure derivatives.

[0127] Depending on the end application, components d) and e) may optionally be included in the composition.

[0128] The present invention also includes a bio-ink composition comprising 0.25% to 10% (w / w) of recombinant bacterial collagen-like protein functionalized with non-terminated olefins, preferably norbornene groups, and / or thiolized recombinant collagen-like protein. Within this concentration range, formulations containing 0.5 to 10% by weight of total recombinant bacterial collagen-like protein are preferred. Total recombinant bacterial collagen-like protein includes any recombinant bacterial collagen-like protein containing functionalizations as described herein.

[0129] Compared to formulations using functionalized animal-derived collagen, the bioink compositions according to the present invention have lower viscosity, thus exerting less shear stress on cells and reducing the chance of nozzle clogging during bioprinting. In the absence of additives, the viscosity of the bioink compositions according to the present invention, when dissolved in water or physiological buffer, can be between 1 and 200 centipoise. In one embodiment, the viscosity of the bioink composition is below 120, below 100, below 80, below 60, below 50, below 40, below 30, below 20, and below 10 centipoise.

[0130] The bio-ink compositions according to the invention can be used to prepare photocrosslinked hydrogels and sponges. Therefore, the invention also relates to a method for producing hydrogels by photocrosslinking functionalized recombinant bacterial collagen-like proteins according to the invention. The bio-ink compositions can undergo photocrosslinking in the presence of photochemical radiation, ultraviolet light, or visible light (VIS) to form transparent and colorless hydrogels. Blue light is particularly used here.

[0131] In a preferred embodiment, the light has a wavelength between 365 nm and 405 nm due to the applied photoinitiator LAP.

[0132] The resulting pH of the bio-ink composition and thus the hydrogel is preferably between 6.5 and 8. The hydrogel obtained by the method according to the invention is also part of this invention.

[0133] The bio-ink composition of the present invention can be dispensed using a bioprinter or poured into a mold for photocuring to form a hydrogel scaffold.

[0134] Another advantage of the bio-ink composition according to the invention is that the resulting hydrogel has significantly more rigid mechanical properties compared to functionalized animal-derived collagen hydrogels, and the rigidity can be tuned (as determined by using the storage modulus measured by rheology) by changing the concentration of the functionalized recombinant bacterial collagen-like protein according to the invention from 0.25 wt% to 10 wt% of the bio-ink composition and / or by selecting different DoFs.

[0135] In one embodiment of the invention, the bioink composition comprises 1.5 to 6% by weight, more preferably 3 to 6% by weight, of the functionalized recombinant bacterial collagen-like protein according to the invention. In one embodiment, the bioink composition further comprises only at least one thiolized recombinant bacterial collagen-like protein according to the invention. In another embodiment, the bioink composition comprises only the recombinant bacterial collagen-like protein functionalized with norbornene groups according to the invention.

[0136] The advantageous mechanical properties of the resulting hydrogel are partly due to the high solubility of the recombinant collagen itself, enabling the formation of high-concentration hydrogels. In contrast, bioinks derived from animal-derived collagen typically contain less than 0.6% (rat tail collagen) due to solubility considerations. The combination of rColN hydrogel and rColS allows for a stiffness of 5.7 kPa, with only 2% total collagen each possessing approximately 50% DoF (degree of functionalization). Using 1% total collagen, a stiffness of only 1.3 kPa is achieved. Using a combination of 5% rColN (77% DoF) and 5% rColS (65%), a stiffness >50 kPa can be achieved. It is assumed that higher concentrations lead to an exponential increase in stiffness. Therefore, hydrogels containing photocrosslinked functionalized recombinant bacterial collagen-like proteins according to the present invention are also part of this invention.

[0137] Additives that can be incorporated into the bio-ink composition as component e) include rheology modifiers, gelation enhancers, and / or bioactive components. These additives can enhance the mechanical, viscoelastic, and biological properties of the bio-ink composition and / or the resulting photocrosslinked hydrogel.

[0138] Self-assembling peptides and biopolymers such as nanocellulose can also be incorporated as component e) into bioink compositions. These molecules enhance gelation and provide additional mechanical properties. Therefore, the present invention also relates to hydrogels comprising photocrosslinked functionalized recombinant bacterial collagen-like proteins according to the invention, the hydrogel further comprising nanocellulose, peptides, or mixtures thereof.

[0139] In another embodiment, the bioink composition includes, as component e), an additional component with a thiol side chain, such as a cysteine-containing protein or drug. Such additives can be used to tailor scaffolds to specific needs.

[0140] In another embodiment, the bio-ink composition comprises glycosaminoglycans as component e), such as chondroitin sulfate, hyaluronic acid, filaments, elastin, keratin, arthropod elastin, myosin, elastin-like peptides, fibrin, fibrinogen, fibronectin, thrombin, chitosan, carbohydrates such as dextran or chitin, growth factors, platelet-rich plasma (PRP), cell-binding peptides, oligonucleotides such as DNA and RNA.

[0141] By adding cells to the bio-ink composition, cells can be encapsulated during photocrosslinking. Cells can also be subsequently incorporated into photocrosslinked hydrogel scaffolds. Whether printed or cast, scaffolds for tissue engineering incorporating the aforementioned hydrogels are also the subject of this invention.

[0142] The resulting photocrosslinked hydrogels support the proliferation of different cell types in vitro. Cells can also be advantageously incorporated into the bioprinting process. Cell viability, proliferation, and diffusion in bioprinted hydrogels are significantly superior to those in bulk-cast hydrogels. Therefore, photocrosslinked hydrogels are particularly suitable as scaffolds for tissue engineering.

[0143] Compared to animal-derived collagen, which is reabsorbed by natural tissues within one month, hydrogels (bioprinted or cast) formed from the bio-ink compositions of the present invention exhibit good in vivo stability for more than three months.

[0144] The bio-ink composition according to the invention can be formulated for various 3D printing or bioprinting technologies, particularly on-demand dripping / jetting and digital light printing / stereolithography. Bio-inks are particularly suitable for on-demand dripping printing due to their low viscosity.

[0145] Bio-inks are also suitable for digital light printing, wherein bio-ink compositions containing 1.5 to 6% by weight of the functionalized recombinant bacterial collagen-like protein according to the invention have been successfully printed into 3D hydrogel constructs.

[0146] Preferably, a bio-ink composition containing 1.5 to 6% by weight of the functionalized recombinant bacterial collagen-like protein according to the invention, more preferably 3 to 6% by weight, can be printed into a 3D hydrogel construct. Combining with compounds containing thiols (such as the other functionalized recombinant bacterial collagen-like proteins described herein, which are recombinant bacterial collagen-like proteins containing at least 60% of the same amino acid sequence as SEQ ID NO:1, wherein the amino acid sequence includes a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:1, and wherein the recombinant collagen-like protein is functionalized with at least one thiol group, as described in this invention) can significantly reduce the concentration required for 3D printing.

[0147] In another aspect, the present invention relates to a method for producing hydrogels by crosslinking, preferably photocrosslinking, the functionalized recombinant bacterial collagen-like proteins or bioink compositions described herein.

[0148] In another aspect, the present invention relates to a hydrogel comprising photocrosslinked functionalized recombinant bacterial collagen-like protein and / or other functionalized recombinant bacterial collagen-like proteins as described herein.

[0149] In one embodiment of the hydrogel of the present invention, the hydrogel further comprises nanocellulose, peptides, or mixtures thereof.

[0150] In another aspect, the present invention relates to scaffolds for tissue engineering comprising hydrogels as described herein.

[0151] The use of the word "a" or "an" can mean "one," but it is also consistent with the meanings of "one or more," "at least one," and "one or more." The use of the term "another" can also refer to one or more. The term "or" is used in the claims to mean "and / or," unless it is explicitly stated that it refers only to alternatives or that the alternatives are mutually exclusive.

[0152] As used herein with respect to any component, “at least one” means the number of chemically distinct molecules or groups, i.e., the number of reference substances of different types, but not the total number of molecules or groups in the composition or compound. For example, “at least one non-terminal olefinic group” means the use of at least one type of non-terminal olefinic group, but two or more different types of non-terminal olefinic groups may also be present, but does not mean that only one or more non-terminal olefinic groups (number) are present.

[0153] As used herein, the words “comprising” (and any form of inclusion, such as “comprising” and “comprising”), “having” (and any form of having, such as “having” and “having”), “including” (and any form of inclusion, such as “comprising” and “comprising”), or “containing” (and any form of containing, such as “comprising” and “comprising”) are inclusive or open-ended and do not exclude additional, unlisted elements or method steps. The term “comprising” also covers and explicitly discloses the terms “consisting of” and “substantially consisting of”. As used herein, the phrase “substantially consisting of” limits the scope of the claim to the specified material or step and the material or step that does not substantially affect the basic and novel features of the claimed invention. As used herein, the phrase “consisting of” excludes any element, step, or component not specified in the claim, except for impurities, for example, those usually associated with elements or limitations.

[0154] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if the order is significant in the particular context. Continuing with this example, explicitly included combinations include repeated forms of one or more items or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that, generally, the number of items or terms in any combination is not limited unless the context explicitly states otherwise.

[0155] As used herein, approximation terms such as, but not limited to, “about,” “approximately,” and “approximately” are those that, when modified, are understood to be not necessarily absolute or perfect, but would be considered sufficiently close to those skilled in the art to guarantee the existence of the specified condition. The extent to which the description may vary will depend on how much change can be made while still allowing those skilled in the art to recognize that the modified feature retains the desired characteristics and capabilities of the unmodified feature. Typically, but in accordance with the foregoing discussion, numerical values ​​modified herein by approximation terms such as “about” may differ from the stated value by ±1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Thus, the term “about” may mean an indication of its value ±5%, preferably an indication of its value ±2%, and most preferably the term “about” precisely means an indication of (±0%).

[0156] The following examples are used to illustrate the present invention and should not be construed as limiting its scope. Example

[0157] Example 1: Preparation of functionalized recombinant bacterial collagen-like proteins with different degrees of functionalization (DoF)

[0158] Norbornene-functionalized CLP (rColN) was synthesized using Vecollan (a recombinant Scl2-based CLP from Evonik Operations GmbH) and 5-norbornene-2-NHS ester (NCA-NHS; BroadPharm; BP-24407). All subsequent steps were performed as sterilely as possible.

[0159] Prepare a clear 4% (w / v) rCol solution in 0.1M HEPES buffer at pH 8.0 overnight on a tracked shaker (450 rpm) at RT (20-25°C). Fill clean, sealable glass vials of varying masses of NCA-NHS, then fill with collagen solution. Stir the covered reaction mixture at room temperature for 24 h. Flocculation disappearance occurs within the reaction time, representing an indicator of reaction progress. Dilute the solution 1:2 with reaction buffer and then dialyze (cellulose tube; cutoff: 12-14 kDa) at RT (20-25°C) for 3 days against alkaline ddH2O (pH ~ 8.0). Change water periodically.

[0160] Every 1-2 hours; at least 3 times a day). The purified product is freeze-dried to produce a white, spongy material, which is then stored at 4°C for further use. Optionally, the product can be aseptically filtered using a 0.2 μm pore size filter before freeze-drying to ensure the material is sterile.

[0161] The degree of functionalization (DoF) is altered by changing the Nor-NHS ratio. In the appended examples, DoMs ranging from 15% to 91% were synthesized.1 H-NMR spectroscopy is used to calculate the degree of DoF.

[0162] The collagen-like protein used contains a tyrosine residue whose independent signal can be used to modulate the overall signal intensity. The degree of functionalization (DoF) is quantified by the peak area ratio of norbornene signals (5.83 to 5.98 ppm and 6.10 to 6.28 ppm and tyrosine signals (6.77 to 6.85 ppm and 7.06 to 7.15 ppm), taking into account the number of hydrogen atoms belonging to the C=C double bond and responsible for each signal. Each collagen chain contains one tyrosine side chain and 23 primary amines (22 lysine side chains and one N-terminal primary amine).

[0163]

[0164] Table 1: Example DoF of different rColN synthesis experiments.

[0165]

[0166] Example 2: Material Safety - Cell Viability of Dissolved and Modified CLP

[0167] 3T3 mouse fibroblasts were used with 1x10 4 Cells / well (1x10) 5 Pre-seed cells / ml (0.1 ml) into clear 96-well plates. Dilute the 20 mg / ml sample stock solution in the culture medium to the following concentrations: 20, 10, 5, 1, 0.1 mg / ml. After incubation for 14–24 hours under cell culture conditions, replace the culture medium with 100 μl of test solution in each well. Perform tests in triplicate for each formulation. As background, use 100 μl of test formulation in the absence of cells. Fresh culture medium with cells is used for negative and positive controls. Expose cells to the test formulation for 24 hours under cell culture conditions. Then, replace the test solution with 100 μl of fresh culture medium + 20 μl of premixed MTS reagent solution (CellTiter) per well. A Queous Non-Radioactive Cell Proliferation Assay (Promega) was performed, resulting in a total volume of 120 μl per well. Ten minutes before adding the MTS solution, 5 μl of Triton X-100 (20% (v / v)) was added to the positive control. The test plate was incubated at 37 °C for 4 hours under culture conditions. The absorbance was then recorded at 490 nm. The mean signal of the positive control was subtracted from each test sample, and the sample signal was normalized on the negative control. The mean and standard deviation of the sample values ​​were calculated.

[0168] Example 3: Preparation of cell-free hydrogels using rColN

[0169] Dissolve 100 mg / ml rColN in 1×PBS until a clear solution is formed. Before the experiment, dissolve 10 mg / ml lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP) in 1×PBS for at least 2 hours. Add 1×PBS and rColN... (aq) and LAP (aq) The samples were mixed in a suitable container and exposed to light with wavelengths ranging from 365 to 405 nm. Different irradiation times were required depending on the applied concentrations of DoF, rColN, and LAP. To demonstrate the importance of concentration, the gelation of the samples in Table 2 was tested.

[0170] Table 2: Gelation assays using rColN and LAP. Total collagen concentration is indicated in the first row. (Left) All formulations used a LAP concentration of 0.3 mg / ml. (Right) All formulations used a LAP concentration of 0.3 mg / ml. 1x PBS was used as the solvent. All samples were irradiated with the same intensity. Successful gelation (√) and insufficient gelation are recorded.

[0171]

[0172] To demonstrate the advantages of using the thiole chemistry of rColN, the material was combined with an equal mass of rColS (such as the thiolated recombinant collagen-like protein described herein).

[0173] Table 3: Gelation assays using the same concentrations of rColN, ColS, and LAP. Total collagen concentration is indicated in the first row. All formulations used a LAP concentration of 0.3 mg / ml. 1x PBS was used as the solvent. All samples were irradiated with the same intensity. Successful gelation (√) and insufficient gelation are recorded.

[0174]

[0175]

[0176] To achieve optimal mixing of the homogeneous solution, the cell-free mixture is vortexed. Due to its low viscosity and lack of proximity to pH limitations or temperature sensitivity, obtaining a homogeneous solution and subsequent hydrogel is considerably easier compared to animal-derived collagen.

[0177] Example 4: Materials suitable for stretching large cells inside hydrogels made of rColN

[0178] 10 mg / ml rColN, 2.5 mg / ml 4-arm-PEG-SH (10 kDa, JenKem Technology USA), and 0.3 mg / ml LAP (phenyl-2,4,6-trimethylbenzoylphosphine) were mixed with HFF cells (P4) in 1x PBS buffer. The mixture was homogenized, and 200 μl sample / well was filled into sterile 8-well slides. Each well contained 1 x 10 5 HFF cells were collected. The preparation was irradiated with light in the wavelength range of 365 nm to 405 nm until the hydrogel solidified. Each sample was mounted with 500 μl of culture medium and incubated for 2 weeks. Two media were used: DMEM high glucose and low serum medium (CnT fibroblast growth medium). The medium was changed every 2-3 days. After 7 and 14 days, HFF cells were used.

[0179] LIVE / DEAD staining (ThermoFisher Scientific) is used to identify live (green) and dead (red) cells. The cell nucleus is...

[0180] Hoechst 33342 (blue) staining.

[0181] Example 5: 3D Bioprinting

[0182] To demonstrate the printability of the material to light processing technology, 50 mg / ml rColN medium with 50% DoM was dissolved in ddH2O containing 0.2% LAP. Printer droplets were successfully generated and gelled using on-demand ink dispensing. Additionally, flat rectangular (…) prints were performed using digital light processing (DLP). Figure 6 ).

[0183] Example 6: Viscosity Measurement

[0184] To better understand the viscosity of the material, rheological measurements were performed using unmodified CLP (rCol). Solution viscosity was measured using an Anton-Paar MCR 502WESP system equipped with a plate-cone extension. The lower plate was flat (stainless steel, Φ50mm, CP50), while the copper cone was angled at 1° to the middle (stainless steel, Φ50mm, CP50-1), with a cutoff of 99 μm. A standard flow profile procedure was applied. The measurement sequence is described below. A 750 μl sample load was used. A stock solution was prepared one day in advance and completely dissolved overnight on a tracked shaker at 25°C. Excess sample was removed with tissue paper. Measurements were performed at 25°C, with data points and shear values ​​from 0.1 to 1000. Data extraction: The mean of data points within the linear range was calculated for each measurement, and the mean of three subsequent measurements was calculated along the standard deviation. Results are shown below. Figure 8 middle.

[0185] Example 7: Rheological Measurement

[0186] Using the desktop ElastoSens from Rheolution Live Sciences TM Bio-devices perform rheological measurements. Non-destructive, non-contact measurements rely on induced vibrations at the bottom of the silicon sample holder, the amplitude of which is recorded by a laser. The harder the formulation, the smaller the amplitude response.

[0187] For photopolymerization, prepare a hydrogel formulation of 2.2 ml. Transfer 2 ml to a calibration sample holder using reverse pipetting. Irradiate the sample with 405 nm (50% lamp power; equivalent to 11.6 mW / cm² according to the provider's power chart) until a stable shear storage modulus (G′) is achieved. Measure stiffness every 10 seconds. Record the hydrogel height through the apparatus, ensuring no significant decrease. Unless otherwise specified, maintain a constant reaction temperature of 25 °C. If the shear storage modulus (G′) recorded using standard stiff mode is less than 500 Pa, repeat the measurement in soft mode with the same formulation using 7 ml, according to the user manual. Results are shown below. Figure 9 middle.

Claims

1. A recombinant bacterial collagen-like protein, preferably comprising an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1, wherein the recombinant collagen-like protein is functionalized with at least one non-terminal olefinic group.

2. The recombinant bacterial collagen-like protein according to claim 1, wherein the amino acid sequence is at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

3. The recombinant bacterial collagen-like protein according to any one of claims 1 or 2, wherein the degree of functionalization with the at least one non-terminal olefinic group ranges from 5% to 100% of the sum of primary amines comprising the N-terminal primary amine group and the primary amine group of the lysine residue of the entire recombinant collagen-like protein.

4. The recombinant bacterial collagen-like protein according to any one of claims 1 to 3, wherein the at least one non-terminal olefinic group is selected from linear, branched or cyclic non-terminal olefinic groups, preferably selected from norbornene or its derivatives, or combinations thereof.

5. A bio-ink composition comprising a) about 0.25 to about 20% by weight of at least one functionalized recombinant bacterial collagen-like protein according to any one of claims 1 to 4; b) Approximately 75% to approximately 99% by weight of an aqueous solvent; c) at least one photoinitiator, in amounts of about 0.01 to about 2% by weight; d) about 0% to about 5% by weight of at least one photocrosslinking polymer or other photocrosslinking peptide; e) about 0 to about 15% by weight of at least one compound selected from additives, rheology modifiers, biopolymers, gelling enhancers, bioactive components, peptides, nanocellulose and / or cells. The condition is that the total weight of all components of the bio-ink is 100%.

6. The bio-ink composition of claim 5, wherein the formulation comprises about 0.125 to about 10% by weight of at least one additional recombinant bacterial collagen-like protein, wherein the additional recombinant bacterial collagen-like protein comprises a functionalization different from that of the functionalized recombinant bacterial collagen-like protein of a).

7. The bio-ink composition of claim 6, wherein the additional functionalized recombinant bacterial collagen-like protein is a recombinant bacterial collagen-like protein comprising at least 60% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:1, and wherein the recombinant collagen-like protein is functionalized with at least one thiol group.

8. The bio-ink composition according to claim 6, wherein the additional functionalized recombinant bacterial collagen-like protein is a recombinant bacterial collagen-like protein comprising at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% of the same amino acid sequence as SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

4.

9. The bio-ink composition according to any one of claims 6 to 8, wherein the degree of functionalization of the additional functionalized recombinant bacterial collagen-like protein ranges from 5% to 100% of the sum of primary amines comprising the N-terminal primary amine and the primary amine group of the lysine residue of the recombinant collagen-like protein.

10. The bio-ink composition of claim 5, wherein the formulation further comprises a thiol molecule, a thiol dimer or a thiol polymer, preferably a multi-arm PEG, such as 4-ArmPEG-SH or dithiothreitol (DTT).

11. A method for producing hydrogels by crosslinking, preferably photocrosslinking, of the functionalized recombinant bacterial collagen-like protein according to any one of claims 1 to 4 or the bio-ink composition according to any one of claims 5 to 10, wherein the bio-ink composition is preferably photocrosslinked using ultraviolet or visible light, preferably blue light.

12. The hydrogel obtained by the method according to claim 11.

13. The hydrogel of claim 12, wherein the hydrogel further comprises nanocellulose, peptides, or mixtures thereof.

14. A scaffold for tissue engineering or tissue regeneration, comprising the hydrogel according to claim 12 or 13.

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