Thiolated 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 the 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 thiol 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 optionally at least one photoinitiator, a method of producing a hydrogel by cross-linking the functionalized recombinant bacterial protein or the bio-ink, and the hydrogel obtained thereby. In addition, a scaffold for tissue engineering comprising the hydrogel.

Description

Technical Field

[0001] This invention relates to bacterial-derived functionalized recombinant collagen-like proteins (CLPs), their use as bio-inks for cell encapsulation, and their use in the preparation of 3D scaffolds, in vitro models, and tissue grafts. Background Technology

[0002] Most bio-inks used in bioprinting are synthetic thermoplastic polymers or animal-derived proteins, such as collagen, gelatin, (meth)acrylamide-functionalized animal-derived collagen, or gelatin methacrylate.

[0003] Collagen-based hydrogels are ideal scaffold materials for cell encapsulation. Their biodegradability and biocompatibility allow for optimal material conditions for bioprinting, while multiple cell-interacting domains (naturally present in collagen) enable cell diffusion and scaffold colonization.

[0004] US2016 / 0051727 discloses collagen-based polymeric materials comprising collagen molecules and / or collagen-derived molecules that are functionalized by adding one or more olefinic unsaturated groups and crosslinked via said groups.

[0005] US2020 / 0179562 discloses curable recombinant human collagen functionalized with methacrylamide, which can be formulated into inks for additive manufacturing and printed into three-dimensional objects.

[0006] US2016 / 0193384 discloses the production of hydrogels by photocrosslinking methacryloyl or acryloyl collagen with synthetic polymers. Such hydrogels can be used as 3D scaffolds and implants, and they are suitable as 3D printing inks for fabricating complex 3D structures or for incorporating cells into structures.

[0007] Photocurable gelatin-based hydrogels have established themselves as powerful bio-inks in tissue engineering due to their excellent biocompatibility, biodegradability, photoresponsiveness, thermal sensitivity, 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 thiolated crosslinking agents have recently gained increasing importance. (etc., 2021). They disclosed a highly reproducible water-based method for synthesizing GelNB, covering a wide range of functionalizations (DoF: 20% to 97%), and its mixing with thiolated gelatin (GelS) to produce ultra-fast curing photoclick hydrogels GelNB / GelS.

[0008] However, the collagens used in the prior art cited above are obtained from human or animal sources. Such human or animal derived collagens are ill-defined due to their natural origin, are sticky and poorly water soluble under physiological conditions. Due to their stickiness, extruding or jetting these collagen mixtures during printing requires considerable force. If cells are co-printed in the same mixture, this leads to greater shear stress, reducing cell viability. The low pH required to solubilize animal derived collagen also makes it challenging to increase the concentration. A further disadvantage due to the source is the quality inconsistency and the risk of disease transmission.

[0009] Collagen-like proteins (CLP) of bacterial origin have interesting mechanical properties, similar to collagen proteins of higher eukaryotes, without the need for the complex maturation steps required for the eukaryotic counterparts. CLP present a common structure: two alpha helices, stabilizing each other, make up a "V domain", followed by a rod-like structural collagen domain (CL). Behind the collagen domain, there is usually a membrane anchor (GPI-like) at the C-terminus of the protein.

[0010] The most relevant CLP for industry is Scl2 of Streptococcus pyogenes. As described in various publications (Lukomski et al., 2002, Brodsky et al., 2009), the current understanding is that the V domain is necessary to fold three Scl2 protein monomers into a triple helix structure in vitro.

[0011] While bioinks based on human or animal derived collagen are widely used, there is still an unmet need for GMP compliant bioinks to facilitate the translation of bio-printed clinical products from the lab to the clinic, in particular water soluble biomaterials that can form hydrogels and thus support cell encapsulation and bio-printing. Needed are properties such as constant quality of production batches, high purity, and good solubility under conditions that provide a suitable environment for encapsulated cells.

[0012] It is therefore an object of the present invention to provide a protein that can be formulated into a bioink and crosslinked into a hydrogel and that at least reduces the disadvantages of the prior art materials, in particular animal derived collagen. SUMMARY

[0013] The present invention thus relates in one aspect to 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 thiol group.

[0014] In one embodiment of the present application, the amino acid sequence is at least 60% identical, preferably at least 70% identical, more preferably at least 80% identical, 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.

[0015] In a further embodiment, the degree of functionalization of the thiol groups of the recombinant bacterial collagen-like protein ranges from 5% to 100% of the sum of the primary amine groups of the entire recombinant collagen-like protein including the primary amine group of the N-terminal primary amine group and the primary amine groups of the lysine residues.

[0016] In another aspect, the present application relates to a bio-ink composition comprising

[0017] a) about 0.125 wt% to about 15 wt% of at least one functionalized recombinant bacterial collagen-like protein according to the present application;

[0018] b) about 75 wt% to about 99 wt% of an aqueous solvent;

[0019] c) about 0 to about 2 wt% of at least one photoinitiator;

[0020] d) about 0 to about 5 wt% of at least one photo-crosslinkable polymer or other photo-crosslinkable peptide;

[0021] e) about 0 to about 15 wt% of at least one compound selected from the group consisting of additives, rheology modifiers, biopolymers, gel strengthening agents, bioactive moieties, peptides, nanocellulose and / or cells;

[0022] with the proviso that the sum of all components of the bio-ink amounts to 100 wt%.

[0023] In one embodiment of the present application, the bio-ink composition further comprises about 0.125 wt% to about 10 wt% of at least one further recombinant bacterial collagen-like protein, wherein said further recombinant bacterial collagen-like protein comprises a different functionalization than the functionalization of the functionalized recombinant bacterial collagen-like protein of a).

[0024] In another embodiment of the present application, said at least one further functionalized recombinant bacterial collagen-like protein is a recombinant bacterial collagen-like protein comprising an amino acid sequence which 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, and wherein the recombinant collagen-like protein is functionalized with at least one alkene group, preferably at least one non-terminal alkene group.

[0025] In one embodiment of the present application, the at least one further functionalized recombinant bacterial collagen-like protein is a recombinant bacterial collagen-like protein comprising an amino acid sequence which is at least 60% identical, preferably at least 70% identical, more preferably at least 80% identical, 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.

[0026] In one embodiment of the bio-ink composition of the present application, the degree of functionalization of the further functionalized recombinant bacterial collagen-like protein ranges from 5% to 100% of the sum of the primary amine groups of the entire recombinant collagen-like protein including the primary amine group of the N-terminal primary amine group and the primary amine groups of the lysine residues.

[0027] In another embodiment of the bio-ink composition of the present application, the at least one non-terminal olefin group is selected from linear, branched or cyclic non-terminal olefins, preferably from norbornene or derivatives thereof, or combinations thereof.

[0028] In a further aspect, the present application relates to a method for producing a hydrogel by cross-linking, preferably photo-cross-linking, of the functionalized recombinant bacterial collagen-like protein or the bio-ink composition described herein.

[0029] In one embodiment of the method of the present application, the bio-ink composition is photo-cross-linked using ultraviolet light or visible light, preferably blue light.

[0030] In yet another aspect, the present application relates to a hydrogel, preferably an injectable hydrogel, obtained by the method according to the present application.

[0031] In one embodiment of the hydrogel of the present application, the hydrogel further comprises nanocellulose, a peptide or a mixture thereof.

[0032] In yet another aspect, the present application relates to a scaffold for tissue engineering comprising the hydrogel described herein. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 : (A) Thiolation reaction scheme of the recombinant bacterial collagen-like protein of the present application, resulting in a recombinant bacterial collagen-like protein comprising thiol groups (rColS). (B) Subsequent disulfide bond formation of rColS for physical gelation.

[0034] Figure 2 : (A) Colorimetric Ellman assay comparison of the functionalized recombinant bacterial collagen-like protein of the present application comprising thiol groups (rColS) with unmodified recombinant bacterial collagen-like protein (CLP). (B) DoF correlation of 1H-NMR spectrum with Ellman assay signal.

[0035] Figure 3Functionalized recombinant bacterial collagen-like proteins (rColS) of the present invention comprising thiol groups and unmodified recombinant bacterial collagen-like proteins (CLP) of the present invention 1 H-NMR example.

[0036] Figure 4 3T3 cell viability after 24h exposure to different concentrations of rCol and rColS. Subsequently, a cell proliferation assay was performed to determine cell viability. The molar ratios applied were equal to the following DoF (%): 1 :0.25 = 15%; 1 :0.5 = 22%; 1 :1 = 39%; 1 :2 = 55% and 1 :5 = 72%.

[0037] Figure 5 Reaction scheme of the photo-induced crosslinking of the functionalized recombinant bacterial collagen-like proteins (rColS) of the present invention comprising thiol groups and the further recombinant bacterial collagen-like proteins (rColN) of the present invention comprising norbornene groups with the photoinitiator LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate).

[0038] Figure 6 Stained human dermal fibroblasts (HDF cells) after 1, 7, 14 and 21 days of cultivation within rColN / rColS hydrogel composites. Cell distribution was observed to be homogenous throughout the hydrogel and cell viability was close to 100% after LIVE / DEAD staining.

[0039] Figure 7 A) Droplets (50 nl) of different shapes printed with the rColN / rColS mixture described herein. B) Droplet-based hydrogel cylinders printed by Drop-on-Demand with 1 % rColN and 1 % rColS formulation. DoF of both materials was ~ 50% ± 5%. 0.03% LAP was used. 1 x PBS was used as solvent. C) Stereolithography printing of CLP cubes made from 1 % rColN and 1 % rColS. DoF of both materials was ~ 50% ± 5%. 0.03% LAP was used.

[0040] Figure 8 A) Hydrogel formulations of 100 mg / ml ColS (DoF of 16%) after 24h storage (4°C) in different buffer systems. From left to right: 1 x PBS, 0.1 M HEPES buffer pH 8.0 and carbonate buffer pH 10. B) 100 mg / ml ColS (DoF of 16% on the left and 54% on the right) was incubated overnight in carbonate buffer pH 10. Subsequently, incubation at 37°C for 10 min liquefied the formulation with a DoF of 16% but not the formulation with a DoF of 54%.​

[0041] Figure 9 Injectability test of ColS hydrogel.

[0042] Figure 10 Gelation reversibility test of 100 mg / ml ColS (DS of 16%).

[0043] Figure 11 The correlation between the concentrations of different rCol and rColS (72% DoF) dissolved overnight in water and the viscosity subsequently measured using an AntonPaar MCR 502WESP system (50 mm plate-cone system, 1° angle; N=3) was investigated. The data presented demonstrate an increase in viscosity due to the introduced thiol modification compared to the unmodified material. This effect is strongly concentration-dependent.

[0044] Figure 12 Example measurement of shear storage modulus using ElastoSens Bio for online measurement.

[0045] Sequence Description

[0046] SEQ ID NO:1 Streptococcus pyogenes collagen-like protein (CLP), full-length protein

[0047] SEQ ID NO:2 Streptococcus pyogenes CLP, truncated 3

[0048] SEQ ID NO:3 Streptococcus pyogenes CLP, truncated 5

[0049] SEQ ID NO:4 Streptococcus pyogenes CLP, without V domain Detailed Implementation

[0050] Collagen-based hydrogels are ideal scaffold materials for cell encapsulation. Their biodegradability and biocompatibility provide optimal material conditions for bioprinting, while multiple cell-interacting domains (naturally present in collagen) allow for cell diffusion and scaffold colonization. Currently available and described collagen-based hydrogels are based on human or animal sources. Such human or animal-derived collagens have several drawbacks, including inconsistent quality, high viscosity, poor water solubility under physiological conditions, and the potential to carry diseases. Due to their viscosity, considerable force is required to extrude or jet viscous collagen mixtures during printing. If cells are co-printed in the same mixture, this results in greater shear stress (thus reducing cell viability). Furthermore, the low pH required to dissolve animal-derived collagen makes increasing concentrations challenging.

[0051] Due to the great need for optimized bio-inks overcoming the above-mentioned drawbacks, the inventors set out to provide a recombinant collagen-like protein of bacterial origin, which is thus a vegan alternative to the collagen materials of animal origin of the prior art, and which can be functionalized and used for the preparation of optimized bio-inks for cell encapsulation and hydrogel production.

[0052] Recombinant collagen-like protein (CLP)

[0053] The industrially most relevant S. pyogenes Scl2 CLP contains a large V-domain, which makes up approximately one third of the entire sequence of Scl2 and hinders the protein from being exported out of the Pichia pastoris host. This requires a complex downstream process involving cell lysis to remove the target protein from the cells. Furthermore, the V-domain itself has pathogenic properties, which need to be removed by protease digestion during the purification process. The use of proteases is rather expensive and needs to be removed in the downstream process. Thus, the production of purified Scl2 starting from the full-length protein including the V-domain requires additional expensive downstream process steps.

[0054] The inventors now surprisingly found that truncated variants of the collagen-like protein, including variants with a truncated V-domain or without any V-domain, lead to an increased production of the collagen-like protein and secretion into the fermentation medium. Even more surprisingly, the truncated variants fold correctly even in the absence of a V-domain.

[0055] Thus, in contrast to what was previously described in the prior art, the V-domain does not seem to be required for the correct folding of the three Scl2 protein monomers into one triple helical structure in vitro, which opens up the possibility to overcome the multiple challenges brought by expressing the full-length Scl2 protein first and then removing the V-domain.

[0056] The present invention thus relates in one aspect to a recombinant bacterial collagen-like protein, which preferably comprises an amino acid sequence which 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 thiol group.

[0057] In one aspect, it is preferred that the recombinant bacterial collagen-like protein comprises a deletion of 38 to 90 amino acids at the N-terminus of the amino acid sequence as set forth in SEQ ID NO: 1, wherein SEQ ID NO: 1 depicts the amino acid sequence of the full-length CLP. It is preferred that the deletion is of 38 to 74 amino acids. This includes the complete deletion of the N-terminal V-domain (comprising 74 amino acids) as well as different truncations of the V-domain of at least 38 amino acids.

[0058] The amino acid sequence of the recombinant bacterial collagen-like protein according to the present application is preferably at least 60% identical to the amino acid sequence 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 versions of Scl2, wherein amino acids have been deleted as follows:

[0059] SEQ ID NO: 2 is based on SEQ ID NO. 1 with deletion of amino acids 13-50 (38 aa)

[0060] SEQ ID NO: 3 is based on SEQ ID NO. 1 with deletion of amino acids 1-74 (75 aa)

[0061] SEQ ID NO: 4 is based on SEQ ID NO. 1 with deletion of amino acids 1-90 (90 aa)

[0062] It is to be understood that the SEQ ID NOs shown herein describe the amino acid sequence as such before functionalization with thiol groups, and not the functionalized recombinant bacterial collagen-like protein.

[0063] In a preferred embodiment, the recombinant bacterial collagen-like protein comprises an amino acid sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. Preferably, the recombinant bacterial collagen-like protein comprises an amino acid sequence which is at least 97% identical, more preferably at least 98% identical, most preferably at least 99% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0064] In a most preferred embodiment, the recombinant bacterial collagen-like protein consists of the amino acid sequence according to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0065] In a further preferred embodiment, other truncated variants of collagen-like proteins from Streptococcus pyogenes are used as a basis to prepare the recombinant bacterial collagen-like protein according to the present application which is functionalized with at least one thiol group.

[0066] The present application therefore also encompasses variants of the recombinant bacterial collagen-like protein according to SEQ ID NOs: 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 at most 5, at most 4, at most 3 or at most 2 amino acid exchanges, insertions and / or deletions.

[0067] In another preferred embodiment of the invention, the variant of the recombinant bacterial collagen-like protein comprises an amino acid sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, preferably at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, more preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, and most preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

[0068] The recombinant bacterial collagen-like protein (CLP) of the present invention can be produced by a method comprising the following steps:

[0069] a) Fermenting bacterial, yeast, or plant host cells expressing CLP in a culture medium, wherein the CLP has 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 contains a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:1.

[0070] b) Accumulate bacterial CLP in the culture medium, from which fermentation broth is obtained.

[0071] c) Separating bacteria, yeast, or plant host cells from the fermentation broth.

[0072] d) Incubate the fermentation broth at no more than 25°C for at least 1 hour for CLP folding.

[0073] e) Optionally purify bacterial CLP by at least one of the following methods: solvent precipitation, tangential flow filtration (TFF), ion exchange chromatography, or reversed-phase chromatography.

[0074] In a preferred embodiment, the CLP folding 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.

[0075] In another preferred embodiment, the CLP folding in step d) is performed for 1 hour to 48 hours, preferably 1 hour to 24 hours.

[0076] In another preferred embodiment, the CLP in step d) is folded at a CLP concentration of at least 1 mg / ml, preferably at least 4 mg / ml.

[0077] In a preferred embodiment, the host cell is a microorganism of the P. pastoris, E. coli, P. putida or C. glutamicum species comprising any polypeptide according to the application.

[0078] In a preferred embodiment, the microorganism is a yeast of the Pichia genus or a bacterial cell, preferably E. coli, Corynebacterium or Brevibactetium.

[0079] The microorganism can be a microorganism in which the nucleotide sequence encoding CLP is present in overexpressing form.

[0080] Functionalization

[0081] The recombinant bacterial collagen-like protein according to the application and described herein above is functionalized with at least one thiol group. The term "thiol group" refers to any organic sulfur compound of the form R-SH, wherein R represents an alkyl group or other organic substituent designed to react with a primary amine. Exemplary thiolating reagents are 2-iminothiolane (2-IT or Traut's reagent), sodium thioparaconate, gamma-thiobutyrolactone and benzoylhomocysteine thiolactone. They are added in a one-step reaction via mechanisms such as Schiff base formation or ring strain release. Thiolating organic molecules with carboxylic acid functionality can also be conjugated to CLP via carbonic acid activation with carbodiimides. Of interest are also N-hydroxysuccinimide activated organic molecules bearing one or more thiol groups.

[0082] More preferably, the recombinant bacterial collagen-like protein is functionalized with a thiolactone group. The term "thiolactone group" refers to an analogue of a lactone, wherein the oxygen atom is replaced by a sulfur atom and the sulfur atom is located within the ring system adjacent to the carbonyl group. The preferred thiolactone is homocysteine thiolactone and derivatives thereof, most preferably N-acetylhomocysteine thiolactone.

[0083] The addition of thiol groups has many advantages compared to known traditional free radical chain polymerization from olefin-containing biomaterials such as GelMA or ColMA (methylacrylated gelatin and collagen) of animal origin, such as faster reaction kinetics, excellent reaction control, concomitant homogeneity within the resulting network, higher conversion of functional groups, less shrinkage during crosslinking leading to less post-polymerization stress (due to the high orthogonality of the reaction), crosslinking reaction not easily inhibited by oxygen, lower radical concentration (i.e. at least one order of magnitude lower than chain growth systems), faster reaction rates (reflected in shorter gel point times), making them more suitable for cell encapsulation and better biomimetic matrices (Van Hoorick, Tytgat et al. 2019).

[0084] The functionalized recombinant bacterial collagen-like protein according to the present application can be prepared by reacting the recombinant bacterial collagen-like protein as described above with a thiol group. Particularly suitable molecules for thiolation are N-hydroxysuccinimide activated organic molecules with one or more thiol hydrogen groups, thioesters, 2-iminothiolane (2-IT or Traut's reagent), sodium thioacetate, heterocyclic thiolactones such as gamma-thiobutyrolactone, N-acetylhomocysteine thiolactone (AcHCT) and benzoylhomocysteine thiolactone.

[0085] In a preferred embodiment, the functionalized recombinant bacterial collagen-like protein according to the present application can be prepared by reacting the recombinant bacterial collagen-like protein as described above with N-acetylhomocysteine thiolactone (AcHCT) in an alkaline carbonate buffer. As Figure 1 The cyclic thioester is conjugated to the primary amine groups of the recombinant bacterial collagen-like protein, as schematically depicted.

[0086] The degree of functionalization of the recombinant bacterial collagen-like protein can be adjusted by adjusting the molar ratio of recombinant bacterial collagen-like protein to thiol group, for example N-acetylhomocysteine thiolactone (AcHCT). After the functionalization step, the mixture is preferably subjected to diafiltration or dialysis to remove the by-products. The degree of functionalization can be determined by known methods, such as trinitrobenzenesulfonic acid assay, Ellman assay, NMR or HPLC-MS.

[0087] The degree of functionalization has an influence on the stiffness of the hydrogel. The higher the degree of functionalization, the stiffer the resulting hydrogel. The stiffness can influence the protein folding and the size of the pores formed in the hydrogel.

[0088] Due to the high solubility of the recombinant bacterial collagen-like protein in neutral and alkaline buffers, an unexpectedly high degree of functionalization of more than 75% can be achieved with minimal damage to the triple helical protein structure.

[0089] In another embodiment, the degree of functionalization of the thiol groups of the recombinant bacterial collagen-like protein ranges from 5% to 100% of the total of the primary amine groups of the entire recombinant collagen-like protein including the primary amine groups of the N-terminal primary amine groups and the primary amine groups of the lysine residues.

[0090] In a preferred embodiment of the present application, the degree of functionalization of the recombinant bacterial collagen-like protein functionalized with thiol groups ranges from 5% to 90% of the total of the primary amine groups of the entire recombinant collagen-like protein including the primary amine groups of the N-terminal primary amine groups and the primary amine groups of the lysine residues.

[0091] In a preferred embodiment of the present application, 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%, at least 95% of the total of the primary amine groups of the recombinant collagen-like protein.

[0092] In a preferred embodiment, the degree of functionalization is from 10% to 90%, from 20% to 80%, from 25% to 75%, from 30% to 70%, from 40% to 60%.

[0093] As described in more detail in Example 1, the degree of functionalization can be modified by adjusting the reaction conditions. By adjusting the molar ratio of AcHCT to recombinant collagen, thiolated recombinant collagen with different degrees of functionalization can be obtained. For example, when the ratio of amine groups of the recombinant collagen to AcHCT was kept at 1 :5, thiolated recombinant collagen with a degree of functionalization of about 72% was synthesized.

[0094] In contrast, collagen of animal origin is usually only about 10% to 20% substituted because extreme reaction conditions denature the triple helical protein structure resulting in gelatin with poor mechanical hardness.

[0095] Another advantage of the thiolated recombinant bacterial collagen-like protein according to the present application is that it is soluble in water, aqueous buffers of neutral or basic pH, in particular at physiologically relevant pH values of 7 to 7.4.

[0096] While the underlying orthogonal step-growth photo-click thiol-ene chemistry is known in the art, the present application provides for the first time a sustainably produced and animal origin free thiolated recombinant CLP (rColS).

[0097] Bio-ink and hydrogel

[0098] For the synthesis of collagen hydrogels, different protein cross-linking methods are known in the literature, but only a few are suitable for 3D bioprinting. This paper provides a light-induced gelation method based on step-growth orthogonal thiol-ene chemistry. Thiolation of CLP results in a new biomaterial (rColS) that is amenable to a fast, signal-induced gelation mechanism without toxic reaction components or by-products during or after gelation.

[0099] Thus, in another aspect, the present application relates to a bio-ink composition comprising

[0100] a) about 0.125% to about 15 wt% of at least one functionalized recombinant bacterial collagen-like protein according to the present application;

[0101] b) about 75 wt% to about 99 wt% of an aqueous solvent;

[0102] c) about 0 to about 2 wt%, preferably about 0.01 wt% to about 2 wt% of at least one photoinitiator;

[0103] d) about 0 to about 5 wt% of at least one photo-crosslinkable polymer or other photo-crosslinkable peptide;

[0104] e) about 0 to about 15 wt% of at least one compound selected from the group consisting of additives, rheology modifiers, biopolymers, gelation enhancers, bioactive moieties, peptides, nanocellulose and / or cells;

[0105] with the proviso that the sum of all components of the bio-ink amounts to 100 wt%.

[0106] The aqueous solvent can be water, any aqueous buffer system, such as 1 x PBS buffer or HEPES buffer, or also a culture medium. Buffer systems and culture media are well known in the prior art and can be selected by the person skilled in the art depending on the desired application.

[0107] In a preferred embodiment, the aqueous solvent is water.

[0108] In the bio-ink composition, the thiol groups can form a polymeric cross-linking network with the functionalized recombinant bacterial collagen-like protein molecules or with other components of the bio-ink composition in the presence of free radicals, anions, nucleophiles or a combination thereof.

[0109] Thus, in one embodiment, the bio-ink composition of the present application thus further comprises about 0.125 wt% to about 10 wt% of at least one further recombinant bacterial collagen-like protein, wherein the further recombinant bacterial collagen-like protein comprises a different functionalization than the functionalization of the functionalized recombinant bacterial collagen-like protein of a).

[0110] In another embodiment of the present application, the at least one further functionalized recombinant bacterial collagen-like protein is a recombinant bacterial collagen-like protein comprising an amino acid sequence which 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, and wherein the recombinant collagen-like protein is functionalized with at least one alkene group, preferably at least one non-terminal alkene group.

[0111] In one embodiment of the present application, the further functionalized recombinant bacterial collagen-like protein is a recombinant bacterial collagen-like protein comprising an amino acid sequence which is at least 60% identical, preferably at least 70% identical, more preferably at least 80% identical, 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.

[0112] In one embodiment of the bio-ink composition of the present application, the degree of functionalization of the further functionalized recombinant bacterial collagen-like protein ranges from 5% to 100% of the total sum of primary amine groups of the entire recombinant collagen-like protein including the primary amine group of the N-terminal primary amine group and the primary amine groups of lysine residues.

[0113] In another embodiment of the bio-ink composition of the present application, the at least one non-terminal alkene group is selected from linear, branched or cyclic non-terminal alkenes, preferably from norbornene or derivatives thereof, or combinations thereof.

[0114] The bio-ink composition of the present application can contain at least one photoinitiator, preferably a free radical photoinitiator. The amount of photoinitiator added to the bio-ink composition ranges from 0 to 2 wt%, preferably from 0.01 wt% to 2 wt% of the total liquid formulation. The photoinitiator is capable of generating free radicals upon irradiation with actinic radiation.

[0115] It is understood that the addition of at least one photo-crosslinkable polymer or other photo-crosslinkable peptide (component d) to the bio-ink composition of the present application is optional, as the functionalized recombinant bacterial collagen-like protein according to the present application can crosslink with itself. This is a unique feature as non-functionalized materials cannot physically gelate.

[0116] In one embodiment, the crosslinking is oxidative crosslinking via the addition of H2O2.

[0117] Preferably, the photo-crosslinkable polymer of component d) is selected from natural (such as hyaluronic acid methacrylate) and synthetic (such as acrylate and methacrylate derivatives of poly(ethylene glycol)) polymers and are mixed into the composition to impart additional biological properties (such as anti-fouling) and to modulate physical properties (such as degradability and swelling behavior in water).

[0118] Synthetic peptides with photo-crosslinkable groups can be incorporated to impart additional biological activities such as cell adhesion, stem cell differentiation and enzymatic degradability. Biological activities are widely described for cell attachment and enzymatic degradation. Biological activities can be tailored via the addition of cell-interaction ligands during photo-crosslinking.

[0119] Photo-polymerization or photo-crosslinking is preferably triggered by exposure to UV or visible light depending on the chosen photoinitiator. A method for producing a hydrogel by photo-crosslinking of the functionalized recombinant bacterial collagen-like protein according to the present invention also forms part of the present invention. Similarly, a method for producing a hydrogel by photo-crosslinking of the bio-ink composition according to the present invention also forms part of the present invention.

[0120] In one embodiment of the method of the present invention, the bio-ink composition is photo-crosslinked using UV light.

[0121] In one embodiment of the method of the present invention, the bio-ink composition is photo-crosslinked using visible light, preferably blue light (380-500 nm). Preferably, light with a wavelength of 390 nm - 410 nm, most preferably 405 nm.

[0122] The bio-ink composition can be formulated by dissolving the functionalized recombinant bacterial collagen-like protein according to the present invention in an aqueous solution, adding a water-soluble photoinitiator such as lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1- one (Irgacure 2959). Other suitable photoinitiators according to the present invention are 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], tris(2,2-bipyridyl)ruthenium(II) dichloride hexahydrate, Eosin Y, Ivocerin, ZnTTP and other Irgacure derivatives.

[0123] Components d) and e) can optionally be included in the composition depending on the final application.

[0124] The present invention also encompasses a bio-ink composition comprising about 1 to 10 wt% of a thiolated recombinant bacterial collagen-like protein and / or a recombinant collagen-like protein functionalized with a non-terminal olefin, preferably a norbornene group.

[0125] The bio-ink composition according to the present application has a lower viscosity compared to formulations using functionalized animal-derived collagen and therefore exerts less shear stress on the cells and has less chance of nozzle clogging during bio-printing. The viscosity of the bio-ink composition according to the present application can be between 1 and 200 centipoise when dissolved in water or a physiological buffer in the absence of additives. In one embodiment, the viscosity of the bio-ink composition is lower than 120 centipoise, lower than 100 centipoise, lower than 80 centipoise, lower than 60 centipoise, lower than 50 centipoise, lower than 40 centipoise, lower than 30 centipoise, lower than 20 centipoise, lower than 10 centipoise.

[0126] The bio-ink composition according to the present application can be applied for the preparation of photocrosslinked hydrogels and sponges. Therefore, the present application also relates to a method for producing a hydrogel by crosslinking, preferably photocrosslinking, the functionalized recombinant bacterial collagen-like protein according to the present application. The bio-ink composition can be photocrosslinked in the presence of actinic radiation, ultraviolet light or visible light (VIS) to form transparent and colorless hydrogels. Here specifically blue light is used.

[0127] In a preferred embodiment, the wavelength of the light applied is between 365 nm and 405 nm due to the applied photoinitiator LAP.

[0128] The resulting pH value of the bio-ink composition and thus of the hydrogel is preferably between 6.5 and 8. Therefore, a hydrogel comprising the photocrosslinked functionalized recombinant bacterial collagen-like protein according to the present application is also part of the present application.

[0129] The bio-ink composition of the present application can be dispensed using a bio-printer or poured into a mold for photocuring to form a hydrogel scaffold.

[0130] Another advantage of the bio-ink composition according to the present application is that the mechanical properties of the resulting hydrogel are significantly stiffer compared to functionalized animal-derived collagen hydrogels and the stiffness (determined by the storage modulus measured by rheology) can be adjusted by varying the concentration of the functionalized recombinant bacterial collagen-like protein according to the present application from 0.125 wt% to 20 wt% (based on the bio-ink composition) and / or by selecting alternative DoFs.

[0131] In one embodiment of the application, the bio-ink composition comprises 0.25 wt% to 15 wt%, 1.0 wt% to 10 wt%, 1.5 wt% to 6 wt%, more preferably 3 wt% to 6 wt% of the functionalized recombinant bacterial collagen-like protein according to the application. In one embodiment, the bio-ink composition comprises only the thiolated recombinant bacterial collagen-like protein according to the application. In a further embodiment, the bio-ink composition further comprises the recombinant bacterial collagen-like protein functionalized with a non-terminal olefin, preferably a norbornene group, according to the application.

[0132] The advantageous mechanical properties of the resulting hydrogels are in part attributed to the higher solubility of the recombinant collagen itself, enabling the formation of hydrogels at high concentrations. In contrast, bio-inks derived from collagen of animal origin are usually less than 0.6% in concentration due to solubility considerations. The stiffness of the recombinant collagen hydrogels (determined by the storage modulus measured by rheology) can be as high as about 6.0 kPa, for example, 10% hydrogel measured at 0.1% strain. Thus, hydrogels comprising the photocrosslinked functionalized recombinant bacterial collagen-like protein according to the application are also part of the present application.

[0133] A further advantage of the thiolated recombinant bacterial-like protein of the present application is that it allows for physical gelation by the application of a temperature change. The inventors could demonstrate that when ColS is dissolved at a certain minimum concentration and stored at 4°C for a certain period of time, an alkali-catalyzed mechanism occurs over time. This gelation is reversible, meaning that the formed gel can be melted by increasing the temperature and re-gel upon cooling. Further experiments showed that an increase in the degree of substitution leads to an increase in the liquefaction temperature. Yet another aspect of the hydrogel of the present application is that it is injectable (see Example 6). This can be used to design hydrogel formulations which, for example:

[0134] a) allow for liquefaction upon oral administration (from the gel at room temperature), or

[0135] b) are initially heated and gel upon injection at 37°C.

[0136] A further adjustable parameter is the concentration of the stock solution. The concentration of the stock solution can have an influence on the gelation properties. Lower concentrations of the stock solution remain in the liquid state for longer storage times at 4°C, while higher concentrations of the stock solution do not. In one embodiment, the concentration of the stock solution is 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, or 100 mg / ml.

[0137] Physical gelation of collagen of animal origin is known from the literature. Irreversibility of collagen of animal origin is also known from the literature. Increasing the temperature to too high values leads to irreversible denaturation of collagen to gelatin. In contrast, for the recombinant collagen of the present invention, heating leads to liquefaction and cooling causes gelation.

[0138] Additives that can be incorporated into the bioink composition as component e) include rheology modifiers, gelation enhancers and / or bioactive moieties. These additives can enhance the mechanical, viscoelastic and biological properties of the bioink composition and / or the resulting photocrosslinked hydrogel.

[0139] In another embodiment, the bioink composition comprises as component e) a glycosaminoglycan, such as chondroitin sulfate, hyaluronic acid; silk, elastin, keratin, resilin, myoxin, elastin-like polypeptide, fibrin, fibrinogen, fibronectin, thrombin, chitosan, a carbohydrate such as dextran or chitin, a growth factor, platelet-rich plasma (PRP), a cell-binding peptide, an oligonucleotide such as DNA and RNA.

[0140] In a further embodiment, the bioink can contain further bioactive ingredients for use as an injectable scaffold as a dermal filler as well as for tissue therapy, such as cartilage, skin or bone repair.

[0141] Self-assembling peptides and biopolymers such as nanocellulose can also be incorporated into the bioink composition as component e). Such molecules enhance gelation and impart additional mechanical properties. Thus, the present invention also relates to a hydrogel comprising the photocrosslinked functionalized recombinant bacterial collagen-like protein according to the present invention, which hydrogel further comprises nanocellulose, a peptide or a mixture thereof.

[0142] Cells can be encapsulated in the photocrosslinking process by adding them to the bioink composition. Cells can also be subsequently incorporated into the photocrosslinked hydrogel scaffold. Whether printed or cast, scaffolds for tissue engineering comprising the above hydrogel are also a subject of the present invention.

[0143] The resulting photocrosslinked hydrogel supports the proliferation of different types of cells in vitro. Cells can also be advantageously incorporated in the bioprinting process. Cell viability, proliferation and spreading in bioprinted hydrogels are significantly superior to bulk cast hydrogels. Thus, photocrosslinked hydrogels are particularly suitable as scaffolds for tissue engineering.

[0144] The hydrogels formed from the bioink composition according to the present invention, whether bioprinted or cast, exhibit good stability in vivo for more than three months, in contrast to collagen of animal origin, which is absorbed by the natural tissue within one month.

[0145] The bio-ink composition according to the present application can be formulated for different 3D printing or bio-printing technologies, in particular drop-on-demand / jetting and digital light printing / stereolithography. Due to the low viscosity, the bio-ink is particularly suitable for drop-on-demand printing.

[0146] The bio-ink is also suitable for digital light printing, wherein a bio-ink composition containing 0.5 wt% to 6 wt% of the functionalized recombinant bacterial collagen-like protein according to the present application has been successfully printed into a 3D hydrogel structure.

[0147] Preferably a bio-ink composition containing 1.5 wt% to 6 wt%, more preferably 3 wt% to 6 wt% of the functionalized recombinant bacterial collagen-like protein according to the present application can be printed into a 3D hydrogel structure.

[0148] In a further aspect, the present application relates to a method for producing a hydrogel by photocrosslinking the functionalized recombinant bacterial collagen-like protein or the bio-ink composition described herein.

[0149] In yet another aspect, the present application relates to a hydrogel obtained by the method according to the present application.

[0150] In one embodiment of the hydrogel of the present application, the hydrogel further comprises nanocellulose, a peptide or a mixture thereof.

[0151] In yet another aspect, the present application relates to a scaffold for tissue engineering comprising the hydrogel described herein.

[0152] The use of the word "a" or "an" can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0153] " At least one" as used herein in reference to any component refers to the number of chemically distinct molecules or groups, i.e., the number of different types of the species referred to, not the total number of molecules or groups in a composition or compound. For example, "at least one thiol group" means that at least one type of thiol group is used, but two or more different types of thiol groups can also be present, but does not mean that only one or more (number) of thiols is present.

[0154] As used herein, the words “comprise” (and any form of inclusion, such as “comprise” and “comprises”), “have” (and any form of having, such as “have” and “has”), “includes” (and any form of inclusion, such as “includes” and “include”), or “contains” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unlisted elements or method steps. The term “comprises” also covers and explicitly discloses the terms “composed of” and “substantially composed of”. The phrase “substantially composed of” as used herein limits the scope of the claims to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. The phrase “composed of” as used herein excludes any element, step, or ingredient not specified in the claims, except for impurities typically associated with the element or limitation.

[0155] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the listed items. 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 includes BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if the order is important in the particular context. Continuing this example, combinations containing repetitions of one or more items or terms are explicitly included, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that there is generally no limit to the number of items or terms in any combination unless the context explicitly states otherwise.

[0156] As used herein, approximate terms such as (but not limited to) “about,” “around,” and “approximately” refer to a condition that, when modified in this way, is not necessarily absolute or perfect, but would be considered by a person skilled in the art to be sufficiently close to guarantee the existence of the specified condition. The extent to which the description may vary will depend on how much variation can be proposed, while still allowing a person skilled in the art to recognize that the modified feature still possesses the desired characteristics and capabilities of the unmodified feature. Typically, but constrained by the foregoing discussion, numerical values ​​modified herein by approximate terms such as “about” may vary from the stated value by ±1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Therefore, the term “about” may mean ±5% of the stated value, preferably ±2% of the stated value, and most preferably, the term “about” means exactly the stated value (±0%).

[0157] The following examples serve to illustrate the present application and are not to be construed as limiting its scope.

[0158] Example

[0159] Example 1 : Preparation of thiolated recombinant collagen-like protein (rColS) with different degrees of functionalization (DoF)

[0160] The functionalized recombinant CLP (rCol) according to the present application was prepared as follows:

[0161] A clear 4% (w / v) rCol solution was prepared in 0.1 M carbonate buffer (pH 10) at room temperature (20 °C). The solution was transferred to a double-necked flask together with 1 mM EDTA (to avoid thiol oxidation by metal ions). The solution was degassed and flushed with N2(3 times each; 1-3 min / step). 160.3 mg / ml DL-N-acetylhomocysteine thiolactone (abbreviated: AcHCT; Sigma & Aldrich; Cat. No. A16602-25G) was dissolved in degassed 0.1 M carbonate buffer (pH 10) at a concentration of 160.3 mg / ml and added via a syringe and septum. After stirring for 3 hours at room temperature (20-25 °C), the reaction mixture was dialyzed against degassed ddH2O (MWCO: 3.5 kDa) for 24 hours at room temperature. The water was changed four times with fresh degassed water. In the first two wash solutions, 1 mM EDTA was added. The purified product was freeze-dried to a white sponge-like material and stored under N2at -80 °C for further use. The DoF was quantified via Ellman assay and H-NMR. 1 H-NMR quantification of DoF.

[0162] Carbonate buffer: 60 ml of a 0.1 M Na2CO3solution was mixed with 40 mL of a 0.1 M NaHCO3solution.

[0163] Table 1: DoF of different rColS synthesis experiments

[0164]

[0165]

[0166] Example 2: Material safety - cell viability of dissolved and modified CLP

[0167] In accordance with the manufacturer protocol, the following were performed: A luminescent cell viability assay was performed to determine the cytotoxic effect of the synthesized collagen derivatives. 3T3 mouse fibroblasts were pre-seeded into white 96-well plates at 1 x 10 4 cells / well (1 x 10 5 cells / ml, 0.1 ml) for luminescence measurements.

[0168] A sample stock solution of 10 mg / ml was diluted in culture medium to the following concentrations: 10, 5, 1, 0.1 mg / ml. After incubation under culture conditions for 14-24 hours, the cell culture medium was replaced by 100 μΐ test solution. Each formulation was tested in triplicate. As background, each formulation was tested without cells with 100 μΐ test. Fresh cell culture medium with cells was used as negative control. Cells were exposed to the test formulation under cell culture conditions for 24 hours. Then, the Reagents Substrate and buffer in a 1 : 1 mixture) and equilibrated for 30 minutes at room temperature (20-25 °C). Reagents were added in 100 μΐ / well. A Tecan Reader Pro 200 orbital was used for 2 minutes of agitation followed by 10 minutes of incubation time. Next, luminescence was measured with an integration time of 0.1 seconds per well. The average signal of the positive control was subtracted from each test sample and the sample signal was normalized against the negative control. The average value of the sample values was calculated as well as the standard deviation.

[0169] Example 3: Bio-ink preparation

[0170] The bio-ink according to the present application was formulated by dissolving the functionalized recombinant bacterial collagen-like protein from Example 1 in phosphate buffered saline, as well as rColN as described herein above. Both homogeneous solutions were mixed and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was added, completely dissolved in phosphate buffered saline. The final concentration of LAP was 0.03% (w / v) of the bio-ink composition (see scheme Figure 5 ) below. Optionally, cells can also be added to the formulation. The formulation was exposed to light with a wavelength of 365-405 nm to induce gelation. Different irradiation times were required depending on the DoF and the concentration of rColN, rColS and LAP applied. The table below shows the test formulations regarding the hydrogel formation. rColN and rColS were applied in the same mass ratio per formulation and different DoF. The DoF was chosen to be similar.

[0171] Table 2: Gelation test using rColN, ColS and LAP. rColN and ColS were always used in the same concentration. All formulations used a LAP concentration of 0.03%. 1 x PBS was used as solvent. All samples were irradiated with the same intensity. Successful gelation and insufficient gelation

[0172]

[0173] Example 4: Material suitability for cell culture against rColN / rColS hydrogels

[0174] In 1 x PBS buffer with 2.5 x 10 6 Formulations of 2.5 mg / ml rColN (48% DoF), 2.5 mg / ml rColS (32% DoF) and 0.3 mg / ml LAP (phenyl-2,4,6-trimethylbenzoylphosphinic acid lithium) were prepared with HDF cells / ml (human dermal fibroblasts). 200 μΐ samples / well were filled into sterile 8-well chamber slides, respectively. The formulations were irradiated with light in the wavelength range of 365 nm - 405 nm until the hydrogel solidified. Each sample was loaded with 200 μΐ medium and incubated for 3 weeks. The medium was exchanged after 2 hours, 24 hours and every 2-3 days thereafter. After 1, 7, 14 and 21 days, live cells (green) and dead cells (red) were determined using LIVE / DEAD staining (ThermoFisher Scientific) Figure 6 ). After 21 days, also deep coding was performed.

[0175] Example 5: 3D bioprinting

[0176] To show the printability of the material, a drop-on-demand printer was used. 10 mg / ml rColN (DoF of 59%) and 10 mg / ml rColS (DoF of 48%) and 0.03% LAP were dissolved together in 1 x PBS (phosphate buffered saline). The homogenized mixture was added to the machine and droplets were printed in different patterns. After light irradiation, the droplets gelled Figure 7 ).

[0177] Example 6: Physical gelation

[0178] 100 mg / ml ColS solutions were prepared in three different buffer systems (1 x PBS, 0.1 M HEPES buffer pH 8.0, carbonate buffer pH 10). After complete dissolution, the formulations were transferred to 2 ml HPLC glass vials and incubated at 4°C for 24 hours. Then the vials were inverted to demonstrate gelation. The DoF (degree of functionalization) influences the liquefaction temperature. While a ColS hydrogel with a DS of 16% liquefied at 37°C, the same hydrogel with a DS of 54% did not Figure 8 ). To show the injectability, a liquid 100 mg / ml ColS formulation was filled into a 1 ml syringe and after incubation at 4°C for 24 hours, the formulation was ejected through a 26G needle Figure 9 ). Finally, the reversibility of gelation was shown by taking a 100 mg / ml ColS solution (DS of 16%) and performing adjacent cycles of gelation and liquefaction Figure 10 ).

[0179] Example 7: Viscosity measurements

[0180] For a better understanding of the viscosity of the material, rheology measurements were performed using unmodified rCol. The solution viscosity was measured using an Anton-Paar MCR 502 WESP system equipped with a plate cone extension. The lower plate was flat (stainless steel,

[0181] CP50), while the copper cone had a 1 ° angle to the middle (stainless steel, CP50-1) with a truncation of 99 pm. The standard flow curve program was applied. The measurement sequence is described below. A sample load of 750 mI was used. The stock solution was prepared one day in advance and left at 20 °C on an orbital shaker overnight for complete dissolution. Excess sample was removed with a paper towel. The measurements were performed at 20 °C data points and a shear of 0.1-1000. Data extraction: the average value of the data points in the linear range of each measurement was calculated and the average value of three consecutive measurements was calculated as well as the standard deviation. The results of the viscosity measurements are depicted in Figure 11 .

[0182] Example 8: Rheology measurements

[0183] Rheology measurements were performed using a desktop ElastoSens TM Bio device from Rheolution Live Sciences. The non-destructive non-contact measurement relies on induced vibrations of the sample holder silicon bottom, the amplitude of which is recorded by a laser. The harder the formulation, the smaller the amplitude response.

[0184] For photopolymerization, 2.2 ml of hydrogel formulation were prepared. 2 ml were transferred to a calibrated sample holder using reverse pipetting. The sample was irradiated with 405 nm (50% lamp power; corresponding to 11.6 mW / cm 2 of light power according to the light power graph provided by the provider) until a stable shear storage modulus (G’) was obtained. The hardness was measured every 10 seconds. The height of the hydrogel was recorded by the device, ensuring no significant drop. If not stated otherwise, the reaction temperature was kept constant at 25 °C. If the shear storage modulus (G’) recorded using the standard stiff mode was below 500 Pa, the measurement was repeated in soft mode using 7 ml of the same formulation according to the user manual. The results are shown in Figure 12 .

Claims

1. A recombinant bacterial collagen-like protein, preferably comprising an amino acid sequence which 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 thiol group.

2. The recombinant bacterial collagen-like protein according to claim 1, wherein the amino acid sequence is at least 60% identical, preferably at least 70% identical, more preferably at least 80% identical, 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 of the thiol groups ranges from 5% to 100% of the sum of primary amine groups of the entire recombinant collagen-like protein including the primary amine group of the N-terminal primary amine group and the primary amine groups of lysine residues.

4. A bio-ink composition comprising a) about 0.125 wt% to about 15 wt% of at least one functionalized recombinant bacterial collagen-like protein according to any one of claims 1 to 3; b) about 75 wt% to about 99 wt% of an aqueous solvent; c) about 0 to about 2 wt% of at least one photoinitiator; d) about 0 to about 5 wt% of at least one photo-crosslinkable polymer or other photo-crosslinkable peptide; e) about 0 to about 15 wt% of at least one compound selected from the group consisting of additives, rheology modifiers, biopolymers, gelation enhancers, bioactive moieties, peptides, nanocellulose and / or cells; with the proviso that the sum of all components of the bio-ink is 100 wt%.

5. The bio-ink composition according to claim 4, wherein the formulation comprises about 0.125 wt% to about 10 wt% of at least one further recombinant bacterial collagen-like protein, wherein the further recombinant bacterial collagen-like protein comprises a functionalization which is different from the functionalization of the functionalized recombinant bacterial collagen-like protein of a).

6. The bio-ink composition according to claim 5, wherein the at least one further functionalized recombinant bacterial collagen-like protein is a recombinant bacterial collagen-like protein comprising an amino acid sequence which 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, and wherein the recombinant collagen-like protein is functionalized with at least one olefin group.

7. The bio-ink composition according to claim 5, wherein the at least one further functionalized recombinant bacterial collagen-like protein is a recombinant bacterial collagen-like protein comprising an amino acid sequence which is at least 60% identical, preferably at least 70% identical, more preferably at least 80% identical, 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. ​ ​ ​ ​ ​ ​ 8. The bio-ink composition according to any one of claims 4 to 7, wherein the degree of functionalization of the further functionalized recombinant bacterial collagen-like protein ranges from 5% to 100% of the total sum of primary amine groups of the entire recombinant collagen-like protein including the N-terminal primary amine group and the primary amine groups of lysine residues.

9. The bio-ink composition according to any one of claims 4 to 8, wherein the at least one alkene group is a non-terminal alkene group selected from a linear, branched or cyclic non-terminal alkene, preferably selected from norbornene or derivatives thereof, or combinations thereof.

10. A method for producing a hydrogel by cross-linking, preferably photo-cross-linking, of the functionalized recombinant bacterial collagen-like protein according to any one of claims 1 to 3 or the bio-ink composition according to any one of claims 4 to 9.

11. The method according to claim 10, characterized in that The bio-ink composition is photo-cross-linked using UV light or visible light, preferably blue light.

12. The hydrogel obtained by the method according to claim 10 or 11.

13. The hydrogel according to claim 12, wherein the hydrogel further comprises nanocellulose, a peptide or a mixture thereof.

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

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