Polypeptide for 3D printing ink

Abstract

The invention relates to a polypeptide which is very suitable for ink for 3D printing. In addition, the invention also relates to polynucleotides encoding the polypeptides of the invention and host cells expressing the polypeptides of the invention. Another embodiment is a method of preparing an ink for 3D printing wherein the ink comprises a polypeptide. Further embodiments relate to methods of making 3D stents and 3D stents obtainable by these methods, especially for use in medical treatment.

Description

Technical Field

[0001] This invention relates to a polypeptide according to claim 1 or 5, which is particularly well-suited for use as ink in 3D printing. Furthermore, the invention relates to a polynucleotide according to claim 7 encoding the polypeptide of the invention and a host cell according to claim 8 expressing the polypeptide of the invention. Another embodiment is a method for preparing ink for 3D printing according to claim 9, wherein the ink comprises a polypeptide. Further embodiments relate to methods for preparing 3D scaffolds according to claims 11 and 13, and 3D scaffolds obtainable by these methods according to claims 15 and 16, including 3D scaffolds for medical use. Background Technology

[0002] In recent years, the 3D printing of 3D scaffolds based on biopolymers has increased significantly. These 3D scaffolds are used either for subsequent seeding of living cells or for directly embedding living cells into a biopolymer matrix during the construction of the 3D scaffold. Among other potential applications, this work also aims to prepare living tissues and organs, such as breast implants composed of living cells that can be implanted in a living body.

[0003] However, not all biopolymers are suitable because some contain amino acid groups or sequences that can elicit an immune response, as the immune system recognizes them as foreign proteins. Furthermore, the biopolymer must be able to be processed into 3D scaffolds at physiologically acceptable temperatures, i.e., dissolved in ink in a liquid state. Additionally, cells must acquire a physiological environment within the biopolymer matrix and adhere well to it to ensure as uniform a distribution as possible while maintaining or developing their biological activity and preserving their differentiated state. Therefore, a biopolymer that closely approximates the properties of the natural extracellular matrix is ​​required. Summary of the Invention

[0004] Therefore, the object of the present invention is to provide a biopolymer that satisfies the above requirements as well as possible.

[0005] Therefore, the present invention provides the polypeptide according to claim 1 or 5, the polynucleotide encoding the polypeptide according to claim 7, and the host cell expressing the polypeptide according to claim 8. A method for preparing 3D printing ink according to claim 9 is also provided, wherein the ink comprises a polypeptide, and the ink according to claim 10. A method for preparing a 3D scaffold according to claims 11 and 13 is also provided, as well as 3D scaffolds obtainable by these methods according to claims 15 and 16. Dependent claims 2 to 4, 6, 12, and 14 relate to preferred embodiments.

[0006] In a first embodiment of the present invention, the present invention relates to a polypeptide comprising a first amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, wherein the polypeptide has another amino acid sequence of length 0 to 200 amino acids at the N-terminus and C-terminus of the first amino acid sequence, respectively. Using the first amino acid sequence enables the provision of a polypeptide that is present in a liquid state in an aqueous solution at a concentration of 10 weight / volume (%w / v) at a temperature of 18°C ​​or higher, preferably 16°C or higher, more preferably 14°C or higher, and most preferably 12°C or higher, thereby making the polypeptide highly suitable for use as a bio-ink for 3D printing. Particularly preferably, the polypeptide of the present invention consists of a first amino acid sequence and corresponding other amino acid sequences located at the N-terminus and C-terminus of the first amino acid sequence. The other amino acid sequence located at the C-terminus of the first amino acid sequence may be the same as or different from the other amino acid sequence located at the N-terminus of the first amino acid sequence.

[0007] In this text, "sequence identity" should be understood as the identical sequence or order of amino acids in a polypeptide. Accordingly, the term "X% sequence identity" should be understood as the amino acid sequences or orders having X% identity.

[0008] "The phrase 'the polypeptide has another amino acid sequence of length 0 to Y amino acids at the N-terminus and C-terminus of the first amino acid sequence' should be understood herein as the first amino acid sequence having at least X% sequence identity with SEQ ID NO: 2 being linked at both ends by peptide bonds to another (peptide) sequence of length 0 to Y amino acids."

[0009] In one specific embodiment of the first embodiment, the first amino acid sequence of the polypeptide of the present invention preferably has at least 92%, more preferably at least 94%, further preferably at least 95%, further preferably at least 96%, further preferably at least 97%, further preferably at least 98%, further preferably at least 99%, and most preferably 100% sequence identity.

[0010] In one specific embodiment of the first embodiment, the polypeptide preferably has another amino acid sequence at the N-terminus and C-terminus of the first amino acid sequence having a length of 0 to 150 amino acids, more preferably 0 to 100 amino acids, even more preferably 0 to 80 amino acids, even more preferably 0 to 60 amino acids, even more preferably 0 to 50 amino acids, even more preferably 0 to 40 amino acids, even more preferably 0 to 30 amino acids, even more preferably 0 to 20 amino acids, and most preferably 0 to 10 amino acids.

[0011] In a second embodiment of the invention, the present invention relates to a polypeptide comprising at least two amino acid sequences, each of which has at least 90% sequence identity with SEQ ID NO: 2. The inventors of this application have discovered that the repetition of said amino acid sequences in the polypeptide of the present invention also results in a polypeptide that is highly suitable for use as a 3D printing bio-ink in terms of gel behavior and viscosity.

[0012] In one specific embodiment of the second embodiment, the amino acid sequences of at least two amino acid sequences of the polypeptide of the present invention preferably have at least 92%, more preferably at least 94%, further preferably at least 95%, further preferably at least 96%, further preferably at least 97%, further preferably at least 98%, further preferably at least 99%, and most preferably 100% sequence identity.

[0013] In one specific embodiment of the second embodiment, the polypeptide preferably has another amino acid sequence with a length of 0 to 100 amino acids, more preferably 0 to 80 amino acids, further preferably 0 to 60 amino acids, further preferably 0 to 50 amino acids, further preferably 0 to 40 amino acids, further preferably 0 to 30 amino acids, further preferably 0 to 20 amino acids, and most preferably 0 to 10 amino acids at the N-terminus and C-terminus of the at least two amino acid sequences, respectively.

[0014] In a third embodiment of the present invention, the present invention relates to a polynucleotide encoding the polypeptide of the present invention according to any of the foregoing embodiments. Preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

[0015] In a fourth embodiment of the invention, the invention relates to a host cell expressing the polypeptide of the invention as described in any of the foregoing embodiments. The host cell preferably does not contain an enzyme complex capable of converting proline to hydroxyproline. The host cell may be a mammalian cell or a microorganism, such as yeast, preferably Pichia pastoris. The enzyme complex is preferably prolyl-4-hydroxylase. Therefore, the polypeptide of the invention is preferably a recombinant polypeptide.

[0016] In a fifth embodiment of the present invention, the present invention relates to a method for preparing ink for 3D printing, wherein a polypeptide is added to a solvent, wherein the polypeptide comprises a first amino acid sequence having at least 85% sequence identity with SEQ ID NO: 2, wherein the polypeptide comprises another amino acid sequence having a length of 0 to 300 amino acids at the N-terminus and C-terminus of the first amino acid sequence, respectively; or comprises at least two amino acid sequences, each of which has at least 85% sequence identity with SEQ ID NO: 2, wherein the polypeptide may be functionalized with crosslinkable groups. Particularly preferably, the polypeptide used in the method of the fifth embodiment of the present invention consists of the first amino acid sequence and another amino acid sequence located at the N-terminus and C-terminus of the first amino acid sequence, respectively. The other amino acid sequence located at the C-terminus of the first amino acid sequence may be the same as or different from the other amino acid sequence located at the N-terminus of the first amino acid sequence.

[0017] According to the present invention, water, water-based buffer systems used in cell culture, or cell culture media can be used as solvents.

[0018] In one specific embodiment of the fifth embodiment, the first amino acid sequence of the polypeptide used in the method of the present invention preferably has at least 87%, more preferably at least 90%, further preferably at least 92%, further preferably at least 94%, further preferably at least 95%, further preferably at least 96%, further preferably at least 97%, further preferably at least 98%, further preferably at least 99%, and most preferably 100% sequence identity.

[0019] In one specific embodiment of the fifth embodiment, the polypeptide used in the method of the present invention preferably has another amino acid sequence with a length of 0 to 250 amino acids, more preferably 0 to 200 amino acids, further preferably 0 to 150 amino acids, further preferably 0 to 100 amino acids, further preferably 0 to 80 amino acids, further preferably 0 to 60 amino acids, further preferably 0 to 50 amino acids, further preferably 0 to 40 amino acids, further preferably 0 to 30 amino acids, further preferably 0 to 20 amino acids, and most preferably 0 to 10 amino acids at the N-terminus and C-terminus of the first amino acid sequence, respectively.

[0020] In a sixth embodiment, the present invention relates to a 3D printing ink comprising a polypeptide having at least 85% sequence identity with SEQ ID NO: 2, wherein the polypeptide has another amino acid sequence of 0 to 300 amino acids at the N-terminus and C-terminus of the first amino acid sequence, respectively; or comprising at least two amino acid sequences, each having at least 85% sequence identity with SEQ ID NO: 2, wherein the polypeptide may be functionalized with crosslinkable groups. Particularly preferably, the polypeptide used in the ink of the present invention consists of the first amino acid sequence and another amino acid sequence located at the N-terminus and C-terminus of the first amino acid sequence, respectively. The other amino acid sequence located at the C-terminus of the first amino acid sequence may be the same as or different from the other amino acid sequence located at the N-terminus of the first amino acid sequence.

[0021] In one specific embodiment of the sixth embodiment, the first amino acid sequence of the polypeptide used in the ink of the present invention preferably has at least 87%, more preferably at least 90%, further preferably at least 92%, further preferably at least 94%, further preferably at least 95%, further preferably at least 96%, further preferably at least 97%, further preferably at least 98%, further preferably at least 99%, and most preferably 100% sequence identity.

[0022] In one specific embodiment of the sixth embodiment, the polypeptide used in the ink of the present invention preferably has another amino acid sequence at the N-terminus and C-terminus of the first amino acid sequence having a length of 0 to 250 amino acids, more preferably 0 to 200 amino acids, even more preferably 0 to 150 amino acids, even more preferably 0 to 100 amino acids, even more preferably 0 to 80 amino acids, even more preferably 0 to 60 amino acids, even more preferably 0 to 50 amino acids, even more preferably 0 to 40 amino acids, even more preferably 0 to 30 amino acids, even more preferably 0 to 20 amino acids, and most preferably 0 to 10 amino acids.

[0023] In a seventh embodiment of the present invention, the present invention relates to a method for preparing a 3D scaffold using ink, preferably using the ink of the sixth embodiment of the present invention, wherein the ink comprises a polypeptide, wherein the polypeptide comprises a first amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2, wherein the polypeptide has another amino acid sequence of length 0 to 350 amino acids at the N-terminus and C-terminus of the first amino acid sequence, respectively; or comprises at least two amino acid sequences, each of which has at least 80% sequence identity with SEQ ID NO: 2, wherein the polypeptide may be functionalized with crosslinkable groups (functionalized polypeptide), and wherein the polypeptide is cured by a 3D printing method.

[0024] In one specific embodiment of the seventh embodiment of the present invention, the polypeptide is preferably functionalized with crosslinkable groups, and light-based molding is preferably used in the 3D printing method.

[0025] The 3D printing or 3D printing method in the sixth and seventh embodiments refers to all 3D printing methods known in the prior art, such as extrusion printing methods, inkjet printing methods, or light-based forming printing methods, such as stereolithography or volumetric / holographic 3D printing. Among these, according to the present invention, light-based forming printing methods are preferred.

[0026] Here, light-based molding preferably refers to the curing of functionalized peptides in the ink by light irradiation, through the cross-linking of cross-linkable groups. Therefore, curing occurs only where sufficient light energy is applied (structured curing). During this process, the peptides cross-link, and curing is achieved in areas irradiated with sufficient light energy. Compared to other bioprinting methods such as extrusion, inkjet, and laser-assisted forward transfer, the exposure method used in this invention, which structures and cures photocrosslinkable peptides to form biocompatible cross-linked polymers, offers a significantly higher printing speed advantage, i.e., the volume of the printed object that can be constructed per unit time. The unique feature of light-based structured curing lies in the combination of high printing speed and high spatial resolution. The performance of this method is based on the laws of optics, and excellent resolution up to the sub-micron level can be achieved by using commercially available optics.

[0027] In volumetric / holographic 3D printing methods, 3D structures are simultaneously solidified by light irradiation, for example by projecting light onto a rotating cylinder or by the intersection of multiple laser beams, such that the light intensity at the intersection of the laser beams is high enough to induce photoinduced crosslinking.

[0028] Stereolithography 3D printing is particularly preferred as the 3D printing method. Stereolithography 3D printing refers to a method of progressively constructing a 3D scaffold structure through point-by-point curing, layer-by-layer curing (intra-layer), or volumetric / holographic curing. According to the present invention, intra-layer curing is preferred. Point-by-point curing preferably uses a laser. In the case of layer-by-layer curing, projection-based exposure or surface exposure is preferred, wherein so-called "digital light processing" is preferred. For speed considerations, according to the present invention, projection-based exposure is preferred.

[0029] In this paper, the term "photocrosslinkable" should be understood as: a functionalized polypeptide that can undergo crosslinking under the influence of electromagnetic radiation and, if necessary, in the presence of a photoinitiator.

[0030] In the eighth embodiment of the present invention, the present invention relates to a method for fabricating a 3D scaffold using an ink containing a polypeptide, preferably using the ink of the sixth embodiment of the present invention, wherein the polypeptide comprises a first amino acid sequence having at least 75% sequence identity with SEQ ID NO: 2, wherein the polypeptide has another amino acid sequence of length 0 to 400 amino acids at the N-terminus and C-terminus of the first amino acid sequence, respectively; or comprises at least two amino acid sequences, each of which has at least 75% sequence identity with SEQ ID NO: 2, wherein the polypeptide is functionalized with crosslinkable groups (functionalized polypeptide), and the polypeptide is cured by stereolithography. Regarding stereolithography, all the contents concerning stereolithography described for the sixth and seventh embodiments also apply to the eighth embodiment.

[0031] In the methods of the seventh and eighth embodiments of the present invention, it is preferable to construct a 3D scaffold using peptides through the following steps: (i) Functionalized peptides in ink are cured by irradiating the ink region where it is desired to be cured or to form a 3D scaffold with electromagnetic radiation (exposure) in a dotted, intralayer or volumetric / holographic manner; wherein, according to the invention, intralayer irradiation is preferred.

[0032] By exposure, functionalized peptides are cross-linked through the cross-linking of functional groups to form cross-linked peptides, which define the spatial structure of the 3D scaffold.

[0033] Here, "in-layer irradiation" should be specifically understood as: when the 3D scaffold of the present invention to be constructed is in ink, it is irradiated with a surface. During this process, only the planar regions where curing is desired in space are irradiated. Planar regions where curing is not desired are not irradiated. In another step (i), the layer of the 3D scaffold of the present invention to be constructed can be introduced into another ink (which may be the same as or different from the ink of the present invention), and the desired regions in that layer can be irradiated accordingly, thereby preparing regions with configurations different from those in the first step (i) within the same layer. Furthermore, the construction of the 3D scaffold of the present invention can also be achieved by repeating step (i) to further superimpose other cured regions with the same or different structures and / or compositions on the already cured regions. In this way, complex 3D scaffolds with different structures can be obtained in each layer.

[0034] In one specific embodiment of the method of the present invention in the seventh and eighth embodiments, the 3D scaffold is preferably constructed by the following steps (preferably in the order listed): (I) Introduce the polypeptide-containing ink (the ink used in the seventh or eighth embodiment) into the reaction vessel. (II) Immerse the support plate on which the 3D scaffold is built into ink, or place it in ink. (III) Irradiate the area where the desired polypeptide will solidify with electromagnetic radiation in a point-like, intralayer, or volumetric / holographic manner. (IV) Cross-linked structures of polypeptides are formed through electromagnetic radiation. (V) Immerse the structure formed in step (IV) together with the carrier plate in another ink (the ink used in the seventh or eighth embodiment or another photocrosslinkable or photopolymerizable ink), or place it in the ink. (VI) Irradiate the area where the other ink is desired to cure with electromagnetic radiation in a dotted, intralayer, or volumetric / holographic manner. (VII) Another polymer or cross-linked structure is formed by electromagnetic radiation in step (VI). (VIII) Repeat steps (V) to (VII) using an ink (the ink used in the seventh or eighth embodiment or another photocrosslinkable or photopolymerizable ink) until a 3D scaffold is constructed.

[0035] The structures formed in the repeated steps are preferably connected to each other by covalent bonds. However, non-covalent bonds, such as those based on physical interactions, can also be used. The entire 3D scaffold to be constructed can be built on a support plate; this support plate can be moved in such a way that the 3D scaffold is sequentially moved into the same or different ink-filled reaction vessels, and in each reaction vessel, photo-based structured curing as described above is performed. For example, according to the invention, all the methods described in EP 3018 531 B1 can be used.

[0036] Therefore, by repeatedly performing ink crosslinking or polymerization steps, layer-by-layer, point-by-point, or volumetric / holographic construction of 3D scaffolds can be achieved. In this process, 3D scaffolds with complex structures can be constructed. Furthermore, inverted and overhanging structures can be formed; this is because even if no crosslinked or polymerized material is placed below a certain point during point-by-point irradiation, and only uncrosslinked or unpolymerized liquid is present, ink crosslinking or polymerization can still occur within that layer or at that point. Ink / peptides located outside that layer or point will not polymerize or crosslink; conversely, only ink / peptides located within that layer or point will undergo crosslinking or polymerization.

[0037] Therefore, in the ninth embodiment of the present invention, the present invention relates to a 3D scaffold that can be obtained by the method of the seventh or eighth embodiment.

[0038] In another embodiment, the present invention relates to a 3D scaffold for medical use according to the invention, or to the use of a 3D scaffold according to the invention in medical applications. Preferably, the 3D scaffold is used as a matrix for living cells. The 3D scaffold can be seeded with living cells after preparation, or the living cells can be present in the ink used to prepare the 3D scaffold. In this way, tissue or in vitro models can be constructed, which can then be used for research or implantation.

[0039] Therefore, the ink used in the sixth embodiment of the present invention or the methods of the seventh and eighth embodiments of the present invention may further contain live cells. Different inks containing the peptides may also be used, but these inks differ in the types of cells used. In this case, the different cell types used can be located at different desired positions within the 3D scaffold.

[0040] Each ink used preferably contains one or more biological cell types. When cross-linking occurs due to electromagnetic radiation, the cells contained in the liquid are embedded together into the 3D scaffold. By using multiple inks (preferably each containing one or more different biological cell types), complex 3D scaffolds, even those with vascular networks, can be constructed. The directional construction of the 3D scaffold of this invention can be achieved using a matrix formed from cross-linked peptides.

[0041] The polypeptides of the present invention, or the polypeptides used according to the present invention, are preferably functionalized with crosslinkable groups, wherein the crosslinkable groups are photoreactive groups.

[0042] In one variant, the crosslinkable group is an acryloyl group, by which the crosslinking of the polypeptide of the present invention or the polypeptide used according to the present invention is achieved. For this purpose, the primary amine in the side chain of the polypeptide of the present invention or the polypeptide used according to the present invention is reacted with an acid anhydride or acyl chloride via a nucleophilic substitution reaction, wherein the latter has a polymerizable double bond. Here, the acid anhydride or acyl chloride of methacrylic acid or carboxynorbornene is particularly preferred.

[0043] The polypeptide is photocrosslinkable by introducing crosslinkable groups, such as acryloyl groups, into the polypeptide of the present invention or the polypeptide used according to the present invention. A crosslinked matrix is ​​prepared by radiation-induced coupling of acryloyl residues between different molecules of the polypeptide of the present invention or the polypeptide used according to the present invention.

[0044] To achieve photocrosslinking via crosslinkable groups, a radical generator (also known as a photoinitiator) is preferably used, which generates radicals at a selected wavelength of the electromagnetic radiation used in the method. Suitable radical generators include, for example, anthrone derivatives (e.g., violanthrone or isoviolanthrone), fluorescein, rubrene, anthracene derivatives, tetraphenylbenzene derivatives, benzanthrone, benzanthronil, eosin, levulinic acid derivatives, phosphine derivatives, monoacylphosphine and diacylphosphine (especially lithium phenyl-2,4,6-trimethylbenzoylphosphine), metallocene, acetophenone, benzophenone, xanthrone, quinone, ketone derivatives, hydroxy ketones, amino ketones, benzoyl peroxide, pyridinium salts, phenylglyoxylate, and / or iodonium salts.

[0045] Furthermore, a substance that prevents photocrosslinking of deeper liquid layers can be added to the ink of the present invention or to the ink used according to the present invention. Therefore, even within the radiation region of the upper focal plane, the liquid solution outside the focal plane remains liquid. This is achieved by the substance absorbing at the wavelength where crosslinking occurs (the crosslinking wavelength). This absorption occurs at the focal plane, thus preventing the crosslinking wavelength from penetrating to deeper layers. Any substance that is absorbent at the desired wavelength is suitable, such as dyes.

[0046] Furthermore, in a variant, the ink of the present invention, or the ink used according to the present invention, may contain a temperature-sensitive gel-forming agent. Reverse temperature-sensitive gel-forming agents are particularly preferred. These gel-forming agents become harder as the temperature increases. By heating the reaction vessel, the reaction liquid solidifies and forms a gel that is initially only metastable. If the ink is not simultaneously photocrosslinked, the metastable gel can be liquefied again and pumped out by subsequently cooling the 3D scaffold. For conventional temperature-sensitive gel-forming agents, the applicable temperature conditions are exactly the opposite. Thus, for example, a support structure can be constructed, thereby forming a cantilever structure. Conversely, if the metastable gel is at least partially irradiated with electromagnetic radiation of a suitable wavelength, photocrosslinking occurs, causing the metastable gel to transform into a stable gel or polymer network in these regions.

[0047] In other words, with the help of temperature-sensitive (especially reverse temperature-sensitive) gelling agents and temperature control of the reaction chamber, overhangs, inverted sections, or cavities can be more easily constructed. In this variant, a liquid structure can still be used as a support.

[0048] Furthermore, temperature gradients can be set so that metastable gels do not form in all regions of the liquid containing temperature-sensitive gel-forming agents (especially inverse temperature-sensitive gel-forming agents). With the help of such gradients, more complex structures can be formed.

[0049] As an alternative or supplement to using gel-forming agents to construct cavities, enzymes can also be used to digest portions of the formed 3D scaffold. The principle is as follows: a 3D scaffold (e.g., a channel system) with cavities / inverted sections is printed as a solid body; this is achieved by filling all cavities with a sacrificial material during the printing process, which can be dissolved subsequently (i.e., after printing) by adding a suitable enzyme. For example, the sacrificial material could be a digestible polymer, which is digested by adding a digestive enzyme. This is a clever strategy for constructing cavities using stereolithography. For example, hyaluronidase (the digestive enzyme) can digest hyaluronic acid (the sacrificial material), thereby creating cavities in the 3D scaffold where hyaluronic acid was originally printed, due to the digestive action of the hyaluronidase.

[0050] Alternatively, to create the cavity / inverted structure, a light-blocking agent can be used in the ink of this invention or in the ink used according to this invention, wherein the light-blocking agent can limit the curing depth of the ink. In this way, curing can also be performed by in-layer irradiation, without having to cure all areas located in the light path in the irradiation depth direction. Compared to the point-by-point method, layer-by-layer curing can significantly improve printing speed.

[0051] In one variation, an optical system is positioned between an electromagnetic radiation source (radiation source) used to generate one or more types of electromagnetic radiation and the reaction vessel. This optical system focuses the electromagnetic radiation onto a corresponding focal plane within the reaction vessel. In another variation, the optical system can be refocused to alter the solidified layer within the reaction vessel. This refocusing can be achieved, for example, by changing the distance between the optical system and the radiation source. In this case, a computer-controlled stepper motor can be used to drive the optical system to perform the corresponding movement. The optical system can be, for example, an optical lens group, or, in cases of particularly simple construction, a single focusing lens. However, alternatively, the solidified point or solidified layer can remain stationary while the 3D scaffold to be constructed moves relative to the electromagnetic radiation.

[0052] For example, the selected irradiation patterns used to fabricate the cross-linked 3D scaffold can be provided by a computer program. Thus, it is conceivable that a user designs the 3D scaffold to be fabricated using a CAD program. The digital object created in this way is then segmented into individual irradiation layers using a suitable computer program. Based on this information, control information for a printer is generated, and the above method is executed using the printer. This control information specifies when which photocrosslinkable ink / liquid must be introduced into the reaction vessel. Furthermore, this control information also specifies when which image of the irradiated layer should be projected onto the corresponding focal plane in the reaction vessel. In this way, the previously computer-designed 3D scaffold can be transformed into a solid 3D scaffold.

[0053] In one variation, the wavelength of the electromagnetic radiation used in the method of the present invention is in the range of 200 nm to 1000 nm (i.e., wavelength between the ultraviolet and infrared regions), more preferably in the range of 350 nm to 800 nm. Substances preferably used as free radical generating agents can be efficiently excited at such wavelengths to generate free radicals, thereby causing crosslinking of polypeptides carrying crosslinkable groups.

[0054] Other suitable wavelengths of electromagnetic radiation used are in the range of 250 nm to 950 nm, particularly 250 nm to 850 nm, particularly 300 nm to 800 nm, particularly 300 nm to 750 nm, particularly 300 nm to 700 nm, particularly 350 nm to 650 nm, and especially particularly 350 nm to 410 nm.

[0055] As can be seen from the foregoing description of the fabrication of the 3D scaffold of the present invention, the 3D printing step of the 3D scaffold can be performed completely automatically, thus requiring no user intervention. This further facilitates the implementation of the method.

[0056] If a 3D scaffold is formed on a substrate, the substrate can be completely removed from the remaining ink in the reaction vessel at the end of the preparation process. The user can then remove the formed 3D scaffold from the substrate.

[0057] Furthermore, the polypeptides of the present invention, or the polypeptides used according to the present invention, preferably do not contain hydroxyproline. In this manner, the polypeptide is a polypeptide existing in a liquid state in an aqueous solution with a concentration of up to 10% (w / v) at a temperature of 12°C or higher.

[0058] Furthermore, the polypeptide of the present invention or the polypeptide used according to the present invention may also be a polypeptide having the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4. Attached Figure Description

[0059] The present invention will now be described in more detail with reference to the following embodiments: Brief description of the attached diagram: Figure 1 A comparison of the temperature-dependent gelation behavior of the polypeptide (ColMP) of the present invention with that of gelatin is shown; Figure 2 The unmodified form of the polypeptide (ColMP) of the present invention is shown. 1 H-NMR; Figure 3 The present invention's polypeptide (ColMP) modified with methacryloyl is shown. 1 H-NMR; Figure 4 The CD spectra of the (ColMP) peptide of the present invention at 5°C, 20°C and 37°C are shown. Figure 5 The CD spectra of gelatin at 5°C, 20°C, and 37°C are shown. Detailed Implementation

[0060] Amino acid analysis: Add 2 to 12 mg of the peptide and 200 µL of a mixture of concentrated hydrochloric acid and trifluoroacetic acid (volume ratio 2:1) to a glass sample tube. Seal the sample tube and hydrolyze at 166 °C for 25 minutes. Then dry completely under vacuum. Dissolve the dried product in 1000 µL of deionized water. The resulting solution is called the hydrolysate.

[0061] For amino acid analysis, a portion of the hydrolysate was diluted so that the diluted solution corresponded to a concentration of 8 to 18 μg (depending on the instrument) of the starting (now hydrolyzed) peptide. Amino acid analysis was performed using a Hitachi 835 high-speed amino acid analyzer under standard conditions for protein hydrolysates. A cation exchange column was used for separation. Amino acid detection was performed spectroscopically using a subsequent ninhydrin reaction. The maximum absorbance of hydroxyproline after colorimetric development was located at 440 nm. The instrument was calibrated using amino acid standards.

[0062] Example 1: Cloning work for preparing the expression construct of the recombinant human polypeptide of the present invention: The gene used to recombinantly prepare the polypeptide of the present invention (SEQ ID NO: 2) is a gene encoding a portion of type I collagen α1 protein. The coding DNA sequence was codon-optimized for expression in yeast (or a typical host organism) and ordered as a product of artificial gene synthesis (Invitrogen). Then, using restriction enzyme digestion and subsequent ligation, the coding sequence was cloned into the reading frame following the α-secretion signal in the pPIC9K vector (Thermo Fisher Scientific). (SEQ ID NO: 5) DNA sequencing confirmed the correct integration of the target gene.

[0063] To purify the structure by immobilized metal ion affinity chromatography (IMAC), the His-6 tag (SEQ ID NO: 4) was inserted into the construct. For this purpose, PCR was performed using primers encoding the His-6 tag at unpaired overhangs, thereby inserting the His-tag-encoding sequence between the polypeptide coding sequence and the stop codon. DNA sequencing confirmed the correct integration of the His-6 tag. (SEQ ID NO: 7)

[0064] Example 2: Transformation of the expression construct into Komagataella phaffii: For transformation, the previously prepared DNA construct was linearized by restriction enzyme digestion and then introduced into the GS115 strain of *Saccharomyces phagnum* (Thermo Fisher Scientific) using electroporation. Positive clones were identified by their growth on selective media.

[0065] Example 3: Expression of the polypeptide of the present invention in a laboratory-scale bioreactor: The recombinant peptides were prepared in a 30 L fed-batch culture method. For this purpose, a 30 L stainless steel bioreactor (Sartorius) was equipped with all necessary sensors (DO, pH, temperature). Additionally, a methanol sensor (Raven Biotech) was installed in the reactor. Fermentation was initiated by inoculating a pre-culture, with the initial optical density (OD) at 600 nm. 600 The concentration was 0.5. During fermentation, the temperature and pH were kept constant at 30℃ and 5, respectively. Dissolved oxygen was maintained above 30% by adjusting the stirrer speed, the gas supply rate, and adding oxygen to the intake air.

[0066] The end of the batch stage is determined by a sudden spike in dissolved oxygen levels when the carbon source in the fermentation medium is depleted. In the subsequent feeding stage, methanol is automatically added based on methanol sensor readings to maintain a constant concentration of 0.5%. Fermentation, as described above, typically concludes after 50-60 hours.

[0067] Example 4: Purification of the polypeptide of the present invention from fermentation broth: For purification, cells were first removed by centrifugation. Then, coarse impurities were removed by filtration through a 1 µm filter, followed by filtration through a 0.45 µm membrane to remove smaller particles and residual cells. The filtrate was then dialyzed using ultrapure water via tangential flow filtration through a 30 kDa membrane until the conductivity reached a constant value. The volume was then reduced by concentration during tangential filtration, and the concentrate was freeze-dried.

[0068] For chromatographic purification, the dried product was dissolved in a suitable flow buffer and further purified by cation exchange chromatography or IMAC. The eluents from both chromatographic methods were then dialyzed through a 30 kDa membrane using tangential flow filtration with ultrapure water until the conductivity reached a constant value. The concentrate was then freeze-dried.

[0069] Example 5: Biocompatibility Test The biocompatibility of the purified peptides was assessed using the MTT assay. For this purpose, L929 cells were seeded in 96-well plates and cultured for 24 hours post-seeding. Then, recombinant peptides at concentrations of 1% and 0.1% (w / v) dissolved in cell culture medium were added to the cells, and the cells were cultured for another 24 hours. For analysis, thiazolyl blue (MTT) at a concentration of 1 mg / mL was added to the cell culture medium, and the cells were incubated at 37°C for 2 hours. The staining solution was then removed, and the formed formazan was dissolved in isopropanol. The metabolic activity of the cells was determined by measuring the absorbance of the isopropanol solution at 570 nm and 650 nm. The measurements were normalized relative to the control group without added substances. The metabolic activity of cells treated with a 1% concentration of the recombinant material solution was typically at least 70% of that of the control group.

[0070] Example 6: Functionalization of the purified product: For 3D printing, the purified peptides were functionalized by introducing methacrylamide groups. To do this, the purified protein was dissolved at 25°C in 0.25 M CB buffer (pH 9) at a concentration of 10% (w / v). The pH was then adjusted to 9. To initiate the reaction, methacrylic anhydride (MAA) (Sigma-Aldrich) was added to the reaction system at a ratio of 0.1 mL per gram of dry protein under continuous stirring. The operation should be carried out in the dark from the addition of MAA. The reaction was then carried out under reflux and condensation for 3 hours. After 3 hours, the reaction was terminated by diluting the reaction solution with CB buffer. The reaction solution was transferred to a dialysis bag with a MWCO of 14 kDa and dialyzed against H2O for 3 days. The dialysate was changed twice daily. After dialysis, the contents of the dialysis bag were filtered through a 0.45 μm filter and freeze-dried under aseptic conditions. The control material (GelMA) was prepared in a similar manner from gelatin (MedellaPro, Gelita).

[0071] Example 7: Rheological analysis of crosslinking properties: To analyze the crosslinking properties, the freeze-dried and functionalized peptide was dissolved in H2O at a concentration of 10% (w / v), with the final concentration of phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP) in the solution being 0.1%. The crosslinking was performed on an Anton-Paar rheometer at a 0.5 mm measurement gap and 37°C, using 1 Hz and 0.1% strain, under UV irradiation (365 nm, 19.95 mW / cm²). 2Before, during, and after the measurement, the crosslinking properties resulting from the introduced methacryloyl groups via a free radical chain reaction (loss modulus and storage modulus) were measured. To allow curing during the measurement, the materials were irradiated with UV light for 180 seconds. GelMA, used as a control material, was measured using the same method. Both materials exhibited a rapid increase in storage modulus (G′) due to the polymerization reaction under UV irradiation.

[0072] Example 8: By 1 H-NMR determination of functionalization: To determine the degree of functionalization, 20 mg of unfunctionalized peptide and 20 mg of functionalized peptide (from Examples 4 and 6) were dissolved in 1 mL of D2O and measured on an Ascend 400 (Bruker) instrument. 1 H-NMR was measured at 400 MHz. The degree of functionalization was determined by the ratio of the integral of the lysine peak in the unmodified and modified recombinant materials, with the phenylalanine peak used as the normalization benchmark. (See modified peptides). Figure 3 Compared to unmodified peptides (see...), Figure 2 The spectrum of the unmodified peptide showed a higher lysine peak at approximately 2.9 ppm. Furthermore, the unmodified peptide lacked the characteristic peaks of the methacryloyl group at approximately 5.3 ppm and approximately 5.6 ppm.

[0073] Example 9: Cell adhesion test using the cross-linked functionalized peptides of the present invention: To test cell adhesion, functionalized peptides were dissolved at a concentration of 10% (w / v) in 0.1% LAP phosphate-buffered saline (PBS). Bio-ink was added to 96-well ultra-low adhesion (ULA) plates and allowed to polymerize completely under light. 8% GelMA ink was used as a positive control, and uncoated wells on the plate were used as negative controls.

[0074] For inoculation, primary human fibroblasts (FS fibroblasts) from the foreskin were added to wells containing polymeric GelMA, wells containing the cross-linked functionalized polypeptide of the present invention (see Example 7), and uncoated wells, and incubated at 37°C for 24 hours.

[0075] Then, live and dead cells were stained with Hoechst 33342, Calcein Red-Orange AM and CellTox Green, and the cells were examined by fluorescence microscopy.

[0076] Compared to GelMA, FS fibroblasts on recombinant peptides showed a lower proportion of dead cells and exhibited better cell adhesion and a more uniform distribution. FS fibroblasts on untreated well plates formed spheroids with a high proportion of dead cells.

[0077] Example 10: 3D bioprinting of the functionalized peptides of the present invention (according to Example 6): For 3D bioprinting, as previously described, functionalized peptides were dissolved in PBS containing 0.1% LAP at concentrations of 10% and 5% (w / v). Then, using the prepared bio-ink, cylinders with a height of 1 mm and a radius of 1 mm were printed on a proprietary multimaterial stereolithography machine (Cellbricks). The material exhibited good printability, with the irradiated area polymerizing within seconds and displaying a clear outline. After 24 hours, FS fibroblasts (20,000 cells / construct) in the construct were examined by fluorescence microscopy using live-dead cell staining, following the method described in the adhesion test. The FS fibroblasts showed adhesion to the printed scaffold material over several days, accompanied by cell migration within the sample material.

[0078] Example 11: Thermogel behavior of the unfunctionalized polypeptide of the present invention (according to Example 4): Based on experience, the functionalized peptides of this invention have low gel points; therefore, the unfunctionalized peptides of this invention were used to measure thermogelation behavior. In this way, it can be confirmed that if the thermogelation behavior of the unfunctionalized peptides is suitable, then the functionalized peptides are also suitable. To measure thermogelation behavior, the unfunctionalized peptides were dissolved in H₂O at 10% (w / v), and losses and storage moduli were measured during a cooling process from 37°C to 4°C at a cooling rate of 0.5°C / min. A 10% gelatin solution was used as a control. Unlike gelatin, the recombinant peptides did not exhibit thermogelation behavior over the ambient temperature range (see [link to relevant documentation]). Figure 1 This behavior can be observed through an increase in storage modulus during cooling. While gelatin shows an increase in storage modulus below about 27°C, recombinant peptides only exhibit this increase below about 12°C, and therefore, unlike gelatin, are liquid at typical operating temperatures for 3D bioprinting (room temperature about 18-22°C) at the same concentration.

[0079] Example 13: Determination of circular dichroism of the unfunctionalized polypeptide of the present invention (according to Example 4): To determine circular dichroism, the recombinant peptide was dissolved in ultrapure water at a concentration of 1 mg / mL and then filtered through a 0.22 μm syringe filter. As a control solution, gelatin was dissolved at a concentration of 1 mg / mL and filtered through a 0.22 μm syringe filter. The circular dichroism of both solutions was measured in the wavelength range of 190 to 290 nm using a J-815 CD spectrometer (Jasco). The graph shows the average of five consecutive measurements. Measurements for both solutions were performed at 5 °C, 20 °C, and 37 °C. (See...) Figure 4 and Figure 5 For gelatin, a typical triple-helix peak appeared at approximately 220 nm at 5°C and 20°C, but was not observed at 37°C. For recombinant peptides, a typical positive peak of the triple helix was not observed at 220 nm at any of the temperatures investigated. This is attributed to the absence or lower degree of prolyl hydroxylation in the peptides of the present invention compared to naturally occurring peptide fragments.

[0080] The following table lists the sequences (SEQ ID NO: 1 to 8) starting from the N-terminus and ending at the C-terminus: Table 1: Protein sequences SEQ ID NO: 1 and 2

[0081]

[0082] Table 2: Protein sequences SEQ ID NO: 3 and 4

[0083]

[0084] Table 3: Nucleotide sequence encoding protein sequence SEQ ID NO: 5: SEQ ID NO: 1

[0085]

[0086]

[0087] Table 4: Nucleotide sequence encoding protein sequence SEQ ID NO: 6: SEQ ID NO: 2

[0088]

[0089] Table 5: Nucleotide sequence encoding protein sequence SEQ ID NO: 7: SEQ ID NO: 3

[0090]

[0091] Table 6: Nucleotide sequence encoding protein sequence SEQ ID NO: 8: SEQ ID NO: 4

[0092]

Claims

1. A polypeptide comprising a first amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, wherein, The polypeptide has another amino acid sequence of 0 to 200 amino acids at the N-terminus and C-terminus of the first amino acid sequence, respectively.

2. The polypeptide according to claim 1, wherein, The first amino acid sequence has at least 95% sequence identity with SEQ ID NO:

2.

3. The polypeptide according to claim 1 or 2, wherein, The polypeptide has another amino acid sequence of 0 to 150 amino acids at the N-terminus and C-terminus of the first amino acid sequence, respectively.

4. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide is substantially free of the amino acid hydroxyproline.

5. A polypeptide comprising at least two amino acid sequences, each of which has at least 90% sequence identity with SEQ ID NO:

2.

6. The polypeptide according to any one of claims 1 to 5, wherein the polypeptide is functionalized with at least one crosslinkable group, wherein, The crosslinkable group may be a photocrosslinkable group.

7. A polynucleotide encoding a polypeptide according to any one of claims 1 to 5.

8. A host cell expressing a polypeptide according to any one of claims 1 to 5.

9. A method for preparing ink for 3D printing, wherein, The polypeptide is added to a solvent, wherein the polypeptide comprises: (a) A first amino acid sequence having at least 85% sequence identity with SEQ ID NO: 2, wherein the polypeptide has another amino acid sequence of length 0 to 300 amino acids at the N-terminus and C-terminus of the first amino acid sequence, respectively; or (b) At least two amino acid sequences, each of which has at least 85% sequence identity with SEQ ID NO:

2. The polypeptide may be functionalized with at least one crosslinkable group, wherein the crosslinkable group may be a photocrosslinkable group.

10. A 3D printing ink comprising a polypeptide, said polypeptide comprising: (a) A first amino acid sequence having at least 85% sequence identity with SEQ ID NO: 2, wherein, The polypeptide has another amino acid sequence of 0 to 300 amino acids at the N-terminus and C-terminus of the first amino acid sequence, respectively. or (b) At least two amino acid sequences, each of which has at least 85% sequence identity with SEQ ID NO:

2. The polypeptide may be functionalized with at least one crosslinkable group, wherein the crosslinkable group may be a photoreactive group.

11. A method for fabricating 3D scaffolds using an ink containing peptides, wherein, The polypeptide comprises: (a) A first amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2, wherein the polypeptide has another amino acid sequence of length 0 to 350 amino acids at the N-terminus and C-terminus of the first amino acid sequence, respectively; or (b) At least two amino acid sequences, each of which has at least 80% sequence identity with SEQ ID NO:

2. The polypeptide can be functionalized with crosslinkable groups, and the polypeptide is cured by 3D printing, preferably by photoinduced chemical reaction.

12. The method according to claim 11, wherein, The polypeptide is functionalized with at least one crosslinkable group, wherein the crosslinkable group may be a photoreactive group, and light-based molding is used in the 3D printing method.

13. A method for fabricating a 3D scaffold using an ink containing peptides, wherein, The polypeptide comprises: (a) A first amino acid sequence having at least 75% sequence identity with SEQ ID NO: 2, wherein the polypeptide has another amino acid sequence of length 0 to 400 amino acids at the N-terminus and C-terminus of the first amino acid sequence, respectively; or (b) At least two amino acid sequences, each of which has at least 75% sequence identity with SEQ ID NO:

2. The polypeptide is functionalized with at least one crosslinkable group, wherein the crosslinkable group may be a photoreactive group, and the polypeptide is cured by stereolithography.

14. The method according to any one of claims 1 to 13, wherein, The ink also contains living cells.

15. A 3D scaffold, which can be obtained by the method according to any one of claims 11 to 14.

16. The 3D scaffold according to claim 15, for medical use.

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