METHOD FOR PRODUCING AN INK FOR 3D PRINTING USING A POLYPEPTIDE BASED ON HUMAN COLLAGEN WITH REDUCED HYDROXYPROLIN CONTENT

Abstract

The invention relates to a method for producing an ink with a polypeptide based on human collagen with a reduced hydroxyproline content, which is ideally suited for 3D printing. Furthermore, the present invention relates to a corresponding ink for 3D printing and its use in medicine. The present invention also relates to a method for producing a 3D scaffold using a 3D printing process with the corresponding ink, as well as to a 3D scaffold.

Description

The present invention relates to a method for producing an ink comprising a human collagen-based polypeptide having a reduced level of ciprofloxacin according to any one of claims 1 to 7, which is excellent in 3D printing. The present invention furthermore relates to a corresponding ink for 3D printing and to its use in medicine according to one of Patent Claims 8 to 10.In recent years, 3D printing of 3D frameworks with a matrix of biopolymers has increased greatly. These 3D frameworks are either used for subsequent colonization of living cells, or the living cells are incorporated directly into the matrix made of the biopolymer during the construction of the 3D framework. In addition to further possible applications, living tissues and organs, such as, for example, breast implants made of living cells, which can be implanted into a living being, are also intended to be produced here.However, no biopolymer is suitable in this case, since some of these biopolymers have sequences and / or groups which can cause an immune reaction, since the immune system recognizes them as foreign proteins, or the biopolymers have sequences of amino acids which the immune system recognizes as foreign. Furthermore, the biopolymers must be processable to form a 3D framework at a physiologically tolerable temperature. Also, biological cells in the biopolymer matrix must find a physiological environment to preserve or develop their biological activity and maintain their state of differentiation. Thus, there is a need for a biopolymer that closely approximates the properties of the natural extracellular matrix.It is therefore the object of the present invention to provide an ink comprising a polypeptide which can be processed in the working temperature range of 3D printing and in which the polypeptide meets the requirements mentioned above as well as possible.To this end, the present invention provides a method for producing an ink for 3D printing according to claim 1, an ink according to claim 8, a method for producing a 3D framework according to claim 11, and a 3D framework according to claim 15. Dependent claims 2 to 7, 9, 10, 12 to 14 and 16 to 18 relate to preferred embodiments.All preferred features mentioned herein with respect to the ink of the method according to the invention for producing an ink are intended here to apply equally to the ink according to the invention, and vice versa. The same also applies to the ink used in the process according to the invention for producing a 3D framework. Likewise, all preferred features mentioned herein with respect to the 3D framework of the method according to the invention for producing a 3D framework should also apply to the 3D framework according to the invention, and vice versa.In one aspect of the invention, the present invention relates to a method for producing an ink for 3D printing, wherein a polypeptide is introduced into a solvent, characterized in that the polypeptide is a human collagen-based polypeptide which has a proportion of hydroxyquinoline of not more than 5%, based on the total number of amino acids of the polypeptide.The present inventors have found that a maximum proportion of 5 % of hydroxypropylproline in the amino acid chain of the polypeptide makes it possible to prepare an ink in which the polypeptide is dissolved. The reason is that such a human collagen-based polypeptide does not form a continuous triple helical structure, so that it is in the liquid state in a 3D pressure working temperature range of preferably 12°C and higher. For the same reason, it is even more preferred that the polypeptide has a proportion of hydroxyquinoline of at most 4%, more preferably at most 3%, even more preferably at most 2%, even more preferably at most 1% and most preferably no hydroxyquinoline, in each case based on the total number of amino acids of the polypeptide. In contrast to naturally occurring collagens, the collagen used according to the invention thus has a lower content of hydroxypropylproline and this is preferably present here as proline.The term "human collagen-based polypeptide" is intended to include all collagens, for example fibrillar (I, II, III, V) and nonfibrillary collagens (IV). Polypeptides comprising or having complete sequences of human collagen, polypeptides comprising or having partial sequences of human collagen, as well as polypeptides comprising or consisting of two or more repeating partial sequences of human collagen, wherein the amino acid sequence of these polypeptides may have at least 80%, preferably at least 90%, more preferably at least 95%, sequence identity with the natural human collagen sequences. This is also intended to encompass the polypeptides mentioned if they are functionalized with at least one crosslinkable group, as described in more detail further below. According to the invention, a polypeptide from or having a subsequence of human collagen is preferred as the human collagen-based polypeptide.Preferably, the human collagen-based polypeptide is a complete or subsequence of collagen I, more preferably the collagen I α-1 chain.By "sequence identity" is meant herein that the sequence of amino acids in the polypeptide is identical. By "X% sequence identity" it is accordingly to be understood that the sequence or order of the amino acid sequence is identical to X%.In a further embodiment of the above-mentioned method according to the invention, it is preferred that the polypeptide is one which is present in the liquid state in an aqueous solution at concentrations of 1 to 10 percent by weight per volume (% w / v) at temperatures of 18° C. and more, preferably of 16° C. and more, more preferably of 14° C. and more, most preferably of 12° C. and more. By "X% w / v" is meant herein that X g polypeptide is contained in 100 ml water. In this way, it is ensured that the ink can be used in the processing region of the 3D print.In a further embodiment of the above-mentioned method according to the invention, it is preferred that the polypeptide is functionalized with at least one crosslinkable group. The crosslinkable group is preferably a photocrosslinkable group. By "photocrosslinkable" is meant herein that the functionalized polypeptide can be crosslinked by exposure to (electromagnetic) radiation and optionally the presence of a photoinitiator.In a further embodiment of the above-mentioned method according to the invention, it is preferred that the polypeptide has a molecular weight in the range from 20 to 200 daltons, more preferably in the range from 25 to 150 daltons, even more preferably 30 to 100 daltons, based on the amino acids of the amino acid sequence.In a further embodiment of the above-mentioned method according to the invention, it is preferred that the ink contains biological cells.A further embodiment according to the invention relates to an ink for 3D printing comprising a polypeptide based on human collagen which has a proportion of hydroxyquinoline of at most 5%, based on the total number of amino acids of the polypeptide. The ink of the present invention is preferably obtainable by the above-mentioned process of the present invention. The method for determining the level of hydroxypropyline is performed using a Hitachi Model 835 High-Speed Amino Acid Analyzer, as described below.The ink is preferably one in which the human collagen-based polypeptide is dissolved in water, preferably in a concentration range as indicated above by the method according to the invention.In a further embodiment according to the invention of the above-mentioned ink, it is preferred that the ink is one for use in medicine, preferably in clinical medicine.A further embodiment of the present invention relates to a method for producing a 3D framework using the ink according to the invention, wherein the polypeptide is cured by means of a 3D printing method. The polypeptide is preferably functionalized with at least one crosslinkable group. In the 3D printing process, the curing of the polypeptide preferably takes place by light-based shaping, preferably by light-induced chemical reaction, in which the crosslinkable groups of the polypeptide are chemically crosslinked with one another.Further, the ink in the method for preparing a 3D framework may contain biological cells. Any biological cells required to mimic a complex biological tissue can be used.A further embodiment of the present invention relates to a 3D framework which has crosslinked polypeptides, wherein the polypeptides are polypeptides based on human collagen, each of which has a proportion of hydroxyquinoline of not more than 5%, based on the total number of amino acids of the respective polypeptide. Here too, the 3D framework according to the invention can have biological cells. The crosslinked polypeptides preferably represent an extracellular matrix for the biological cells in which the biological cells are embedded. Preferably, the 3D framework of the invention is one obtainable by the process of the invention for producing a 3D framework. The 3D scaffold according to the invention can be used in medicine, for example for constructing replacement tissue structures.Suitable solvents for the peptides in the ink according to the invention or used according to the invention are water or water-based buffer systems from cell culture or else cell culture media.In a particularly preferred embodiment, the polypeptide used according to the invention is a polypeptide comprising a first amino acid sequence with at least 70% sequence identity to SEQ ID No. 2, wherein the polypeptide has a further amino acid sequence with a length of 0 to 200 amino acids at the first amino acid sequence both at its N-terminal end and at its C-terminal end. Use of the first amino acid sequence allows the provision of a polypeptide that is in the liquid state in an aqueous solution at concentrations of 10 weight percent per volume (% w / v) at temperatures of 12°C and higher, such that the polypeptide is well suited for use in biotinates for 3D printing. Particularly preferably, the polypeptide used according to the invention consists of the first amino acid sequence and the respective further amino acid sequence at the N-terminal end and the C-terminal end of the first amino acid sequence. The further amino acid sequence at the C-terminal end of the first amino acid sequence may be the same as or different from the further amino acid sequence at the N-terminal end of the first amino acid sequence. As already mentioned, the polypeptides used according to the invention are preferably present functionalized with at least one crosslinkable group, and in the 3D printing, light-based shaping is preferably used.By "sequence identity" is meant herein that the sequence of amino acids in the polypeptide is identical. By "X% sequence identity" it is accordingly to be understood that the sequence or sequence of the amino acid sequence is identical to X%.By "the polypeptide at the first amino acid sequence has a further amino acid sequence with a length of 0 to Y amino acids both at its N-terminal end and at its C-terminal end" is meant herein that the first amino acid sequence with at least X% sequence identity to SEQ ID NO:2 is linked at both ends via a peptide bond to a further (peptide) sequence with a length of 0 to Y amino acids.In one embodiment of the first embodiment, the first amino acid sequence of the polypeptide according to the invention preferably has at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 93%, even more preferably at least 95%, even more preferably at least 98% and most preferably 100% sequence identity.In one embodiment, the polypeptide used according to the invention preferably has at the first amino acid sequence both at its N-terminal end and at its C-terminal end a further amino acid sequence with 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.Furthermore, the polypeptide used according to the invention can also be one having a sequence according to SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 4, or can result from these by cleavage of an initial or end sequence.The polypeptide used in the present invention is preferably encoded by a polynucleotide. For this purpose, it is preferred that the polynucleotide comprises a nucleotide sequence according to SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. For this purpose, a host cell is preferably used which expresses the polypeptide used according to the invention. The host cell is preferably one free of an enzyme complex capable of converting proline to hydroxypropylproline as mentioned above. The host cell may be a mammalian cell or a microorganism, for example yeast such as Pichia pastoris. The enzyme complex is preferably prolyl-4-hydroxylase. The polypeptide used according to the invention is therefore preferably a recombinantly produced polypeptide.Herein, 3D printing or 3D printing methods are to be understood as meaning all 3D printing methods known in the prior art, such as e.g. extrusion printing methods, ink jet printing methods or printing methods under light-based shaping, such as stereolithography or volumetric / holographic 3D printing, wherein printing methods under light-based shaping are preferred according to the invention.Light-based shaping is preferably understood herein to mean that the functionalized polypeptide is cured from the ink by light irradiation by linking the crosslinkable groups to one another, preferably by chemical reaction. It is therefore cured only at those locations where sufficient light energy is also irradiated (structuring curing). The polypeptides are crosslinked among one another and the polypeptide cures in the region in which sufficient light energy is radiated in. An exposure process with structuring curing of the photocrosslinkable polypeptide to form a biocompatible crosslinked polymer-as used according to the invention-has the advantage over other biological printing processes, such as the extrusion process, the ink jet process and the laser-supported forward transfer process of a superior printing speed, i.e. a large volume of the object to be printed per unit time can be built up. Unique to light-based structured curing is the combination of high printing speed and high spatial resolution. The performance of the method is based on optical laws and superior resolution to the sub-micron range is possible through the use of commercially available optics.Under a volumetric / holographic 3D printing method, a 3D structure is cured simultaneously by irradiation with light, for example by light projection onto a rotating cylinder, or by crossing a plurality of laser beams, as a result of which the light intensity is high enough for the light-induced crosslinking at the points of the crossing of the laser beams.Particularly preferably, a 3D stereolithographically printing method is used as the 3D printing method. A 3D stereolithographically printing method is understood to mean one in which the structure of the 3D framework is produced gradually by point, layer-by-layer (in a layer) or volumetric / holographic curing, wherein curing within a layer is preferred according to the invention. In the case of point curing, a laser is preferably used. During the layer-by-layer curing, preferably projection-based or extensive irradiation is carried out, wherein the so-called "digital light processing" is preferably used. For speed reasons, a projection-based exposure method is preferred according to the invention.By "photocrosslinkable" is meant herein that the functionalized polypeptide can be cross-linked by exposure to electromagnetic radiation and optionally the presence of a photoinitiator.In the methods of the invention for the production of 3D frameworks, it is preferred that a 3D framework is constructed from the polypeptide by the following step:(i) Curing of the functionalized polypeptide from the ink by irradiation of electromagnetic radiation (exposure) at points, in a layer or volumetrically / holographically in the region of the ink in which curing or building up of the 3D framework is desired, wherein irradiation in a layer is preferred according to the invention.By exposure, a crosslinked polypeptide is obtained from the functionalized polypeptide by crosslinking the functional groups, which specifies a spatial structure of the 3D framework.Here, the irradiation in a layer is to be understood in particular as meaning that the 3D framework according to the invention to be constructed is irradiated two-dimensionally while it is located in the ink. Only those areas in the area where hardening is desired spatially are irradiated. In this case, regions of the surface in which curing is not desired cannot be irradiated. In a further step (i), however, it is then also possible to produce a region having a different structure than in the first step (i) in the same layer of the 3D framework according to the invention to be constructed by introducing the layer into a further ink (the same as or different from the ink according to the invention) and correspondingly irradiating it into the desired region of the layer. Furthermore, the 3D framework according to the invention can also be constructed by applying further cured regions of the same or different structure and / or composition to the already cured regions by repeating step (i). In this way, a complex 3D framework having different structures in each layer can be obtained.In one configuration of the method according to the invention for producing a 3D framework, it is preferred that the 3D framework is constructed by the following steps (preferably in the sequence indicated):(I) introducing said ink containing said polypeptide into a reaction vessel;(II) immersing or providing a support plate on which the 3D skeleton is to be mounted in said ink;(III) Irradiation of electromagnetic radiation at points, in a layer or volumetrically / holographically in the regions in which curing of said polypeptide is desired;(IV) generating a crosslinked structure of the polypeptide by the electromagnetic radiation,(V) dipping or presence of the structure formed in step (IV) on the support plate in another ink (said ink or another photocrosslinkable or photopolymerizable ink);(VI) Irradiation of electromagnetic radiation at points, in a layer or volumetrically / holographically in the regions in which curing of the further ink is desired,(VII) creating another polymerized or crosslinked structure by the electromagnetic radiation in step (VI),(VIII) Repeating steps (V) to (VII) with each ink (either said ink or another photocrosslinkable or photopolymerizable ink) until the 3D backbone is formed.The structures produced in the repeated steps are preferably connected to one another by covalent bonds. However, non-covalent bonds, for example based on physical interactions, are also possible. The entire 3D framework to be built up can be built up on a carrier plate which can be moved in such a way that the 3D framework is moved successively into identical or different reaction vessels filled with ink and the light-based, structuring curing is carried out there in each case, as has already been described above. All the processes mentioned in EP 3 018 531 B1 can be used, for example, according to the invention.By repeating the process steps in which crosslinking or polymerization of the inks takes place, a layer-by-layer, point-by-point or volumetric / holographic construction of the 3D framework is consequently achieved. It is possible to construct a 3D framework with a complex structure. Moreover, undercuts and overhanging structures can also be formed, since crosslinking or polymerization of the ink in a specific layer or else at a specific point during the point irradiation can also take place if no already crosslinked or polymerized material is arranged underneath it, but rather only liquid which has not yet been crosslinked or polymerized. Polymerization or crosslinking of an ink / polypeptide present outside the layer or dot is eliminated; rather, only that ink / polypeptide which is within the layer or dot is crosslinked or polymerized.For use of the 3D framework of the present invention in medicine, it is preferable that the 3D framework is used as a matrix for living cells. The 3D framework can either be colonized with living cells after its preparation, or the living cells are in the ink from which the 3D framework is prepared. In this way, tissues or in vitromodels can be built up, which can then be used in research or even implanted.The ink according to the invention or used according to the invention can consequently additionally have live cells. In this case, it is also possible for different inks to be used with the polypeptide mentioned, which however differ with regard to the cell types used. In this case, the various cell types used can sit at different, desired positions within the 3D framework.The inks used preferably each contain one or more biological cell types. When crosslinking occurs due to irradiation of the electromagnetic radiation, the cells contained in the liquid are embedded in the 3D framework. By using multiple inks, preferably with one or more different biological cell types, a complex 3D framework can be constructed that also has a blood vessel structural network. The use of a matrix of the crosslinked polypeptide thereby enables the directed construction of the 3D framework according to the invention.The polypeptide used according to the invention is preferably functionalized with a crosslinkable group which is a photoreactive group. In one variant, the crosslinkable group is an acrylic group by means of which the crosslinking of the polypeptide used according to the invention is effected. For this purpose, the primary amines in the side chains of the polypeptide used according to the invention are preferably reacted via a nucleophilic substitution reaction with an anhydride or an acid chloride, the latter having a polymerisable double bond. Particular preference is given here to using an anhydride or acid chloride of methacrylic acid or of carboxynorbornene.By incorporating a crosslinkable group, for example an acrylic group, into the polypeptide used in the present invention, it is made photocrosslinkable. Radiation-induced coupling of the acrylic residues between different molecules of the polypeptide used according to the invention produces a cross-linked matrix.In order that photocrosslinking via the crosslinkable group can be achieved, a radical generator (a so-called photoinitiator) is preferably used, which generates radicals at a selected wavelength of the electromagnetic radiation used in the context of the process. Suitable radical formers are, for example, anthrone derivatives, such as, for example, violanthrone or isoviolanthrone, fluorescein, rubrene, anthracene derivatives, tetrazene derivatives, benzanthrone, benzanthronil, eosin, levolic acid derivatives, phosphine derivatives, mono- and bis-acyl phosphines, in particular lithium phenyl-2,4,6-trimethylbenzoylphosphinate, metallocenes, acetophenones, benzophenones, xanthones, quinones, ketone derivatives, hydroxy ketones, amino ketones, benzoyl peroxides, pyridine salts, phenyl glyoxylates and / or iodonium salts.In addition, a substance which prevents photocrosslinking of deeper, liquid layers can be added to the ink used according to the invention. Thus, liquid solution remains liquid outside the focal plane, even if it is located in the irradiation region of the focal plane lying above it. This functions by absorbing the substance at the wavelength at which the crosslinking takes place (crosslinking wavelength). The interception takes place in the focal plane, so that no penetration of the crosslinking wavelength into deeper layers is possible. Suitable substances are all substances which absorb at the desired wavelength, such as dyes.In addition, it is possible in one variant for the ink used according to the invention to have a temperature-sensitive gelling agent. In particular, the use of an inversely temperature-sensitive (also referred to as reverse temperature-sensitive) gelling agent is provided. Such a gelling agent becomes more solid with increasing temperature. By heating the reaction vessel, the reaction liquid solidifies and initially forms only a metastable gel. If the ink is not simultaneously photocrosslinked, the metastable gel can be liquefied again and pumped off by subsequent cooling of the 3D backbone. In the case of conventional temperature-sensitive gelling agents, the temperature conditions to be used are exactly the reverse. For example, if necessary, a supporting structure can be created, so that suspended structures can be produced. If, on the other hand, the metastable gel is at least partially irradiated with electromagnetic radiation at a suitable wavelength, photocrosslinking occurs, so that the metastable gel is converted into a stable gel or polymer network in these regions.In other words, the temperature-sensitive, in particular inversely temperature-sensitive, gelling agent and a temperature control of the reaction space make it even easier to work with suspended portions and undercuts or cavities. In this variant, too, it is possible to work with liquid structures as support.It is also possible to provide a temperature gradient so that a metastable gel does not take place in all regions of the liquid to which the temperature-sensitive, in particular inversely temperature-sensitive, gelling agent has been added. With the aid of such a gradient, even more complex structures can be produced.Alternatively or in addition to the formation of voids via the use of a gelling agent, enzymes can also be used for digestion of parts of the 3D framework formed. The principle is as follows: a 3D framework with cavities / undercuts (e.g. a channel system) is printed as a solid body in that all cavities are filled with a sacrificial material during printing, which can be dissolved later (i.e. after completion of the printing) by administration of the correct enzyme. The sacrificial material is, for example, a digestible polymer which is digested by addition of a digestive enzyme. This is an elegant strategy for creating voids with stereolithographically printing techniques. For example, a morpholinidase (digestive enzyme) can digest hyaluronic acid (sacrificial material) so that a cavity is formed at the site in the 3D framework in which hyaluronic acid has been printed by morpholinidase digestion.Alternatively, a photoblocker can also be used in the ink according to the invention or the ink used according to the invention to create a cavity / undercut, wherein the photoblocker restricts the curing depth of the ink. In this way, the curing can also be effected by irradiation in a layer without curing in the irradiation depth all regions which lie in the beam path. The possibility of layer-by-layer curing increases the printing speed in comparison with point processes immensely.In one variant, an optical system is arranged between a source for the electromagnetic radiation (radiation source), which serves to generate the one and / or the further electromagnetic radiation, and the reaction vessel, which optical system serves to focus the electromagnetic radiation onto the respective focal plane in the reaction vessel. In this case, in one variant, it is provided that refocusing of this optical system can take place in order to change the layer of curing within the reaction vessel. Such refocusing can be achieved, for example, by changing the distance of the optical system from the radiation source. A computer-controlled stepping motor can be provided in this case in order to impart a corresponding movement of the optical system. The optical system can be, for example, a system of optical lenses or-in a case which is particularly simple in terms of design-a single focusing lens. Alternatively, however, the point or the layer of curing can also be static and the 3D framework to be built up can be moved relative to the electromagnetic radiation.The respectively selected irradiation pattern for producing the networked 3D framework can be provided, for example, by a computer program. It is thus conceivable for a user to create the 3D framework to be produced by means of a CAD program. The digital object produced in this way is then cut by a suitable computer program into individual irradiation planes. For this information, control information is created for a printer, by means of which the described method is carried out. This control information specifies when which photocrosslinkable ink / liquid must be introduced into the reaction vessel. Furthermore, this control information specifies when which image of an irradiation plane is to be projected onto the respective focal plane in the reaction vessel. In this way, the 3D framework previously created on the computer can then be converted into a real 3D framework.In one variant, the electromagnetic radiation used in the methods according to the invention has a wavelength in the range from 200 nm to 1000 nm (i.e. a wavelength which lies between the UV range and the infrared range), more preferably in the range from 350 nm and 800 nm. With such wavelengths, the substances preferably used as free radical formers can be excited particularly well, so that free radicals are formed in order to enable crosslinking of the polypeptide carrying the crosslinkable groups.Further suitable wavelengths of the electromagnetic radiation used are in the range from 250 nm to 950 nm, in particular from 250 nm to 850 nm, in particular from 300 nm to 800 nm, in particular from 300 nm to 750 nm, in particular from 300 nm to 700 nm, in particular from 350 nm to 650 nm and very particularly from 350 nm to 410 nm.As can be seen from the previous illustration of the production of the 3D scaffold according to the invention, the step of 3D printing the 3D scaffold can be carried out in a completely automated manner, so that an intervention by a user is not required. This additionally facilitates the application of the method.When the 3D framework is produced on a carrier plate, this carrier plate can be completely lifted out of the ink still remaining in the reaction vessel after the end of the production process. The 3D framework produced can then be removed from the carrier plate by the user.The present invention will now be illustrated in more detail by the following examples:FIGS. : Figure 1 shows temperature dependent gelling behaviour of the polypeptide according to the invention (ColMP) compared to gelatin; FIG. 2 shows a 1 H-NMR of the inventive polyeptide (ColMP) in unmodified form; Figure 3 shows a 1 H-NMR of the polypeptide of the invention (ColMP) modified with methacryloyl groups; Figure 4 shows a CD spectrum of the (ColMP) polypeptide of the present invention at 5°C, 20°C and 37°C; Fig. 5 shows a CD spectrum of gelatin at 5°C, 20°C and 37°C.Amino acid analysis:In a glass sample tube, 2 to 12 mg of polypeptide and 200 μL of a mixture of concentrated hydrochloric acid and trifluoroacetic acid (2:1 by volume) are added. The sample tube is sealed and hydrolyzed at 166°C for 25 min. The whole is then dried under vacuum. The dry product is dissolved in 1000 μL demineralized water. The resulting solution is referred to as hydrolyzate.For amino acid analysis, a portion of the hydrolysate is diluted so that the diluted solution corresponds to a concentration of 8 to 18 μg of the starting, now hydrolysed, polypeptide (depending on the instrument). Amino acid analysis is carried out using a Hitachi Model 835 High-Speed Amino Acid Analyzer under standard conditions for protein hydrolysates. A cation exchange column is used for separation. The detection of the amino acids is carried out spectroscopically by means of subsequent use of a ninhydrin reaction. The absorption maximum of the colored hydroxypropyline is 440 nm. An amino acid standard was used to calibrate the instrument.Example 1: Cloning Works for Producing an Expression Construct for Recombinant Human Polypeptides of the InventionThe gene for recombinant production of the polypeptide of the present invention (SEQ ID NO:2) is one encoding a part of the human collagen I alpha 1 protein. The coding DNA sequence was codon optimized for expression in yeast (or general organism) and ordered as a product of artificial gene synthesis (Invitrogen). The coding sequence was then cloned by restriction digestion followed by ligation in frame behind the α-secretion signal of the pPIC9K vector (Thermo Fisher Scientific). (SEQ ID NO:5) Correct integration of the target gene was confirmed by DNA sequencing.For purification by immobilized metal ion chromatography (IMAC), a His-6 tag was inserted into the construct described above (SEQ ID NO:4). For this, by PCR with a primer encoding the His-6 tag on a non-binding overhang, the His-tag encoding sequence was inserted between the polypeptide encoding sequence and the stop codon. Correct integration of the His-6 tag was confirmed by DNA sequencing. (SEQ ID NO:7)Example 2: Transformation of the expression construct into Komagataella phaffii:For the transformation, the previously generated DNA construct was linearized by restriction digestion and then introduced into the GS115 Komagataella phaffiistrain (Thermo Fisher Scientific) by means of electroporation. Positive clones were identified by growth on selection medium.Example 3: Laboratory scale bioreactor expression of the polypeptide of the invention:The recombinant polypeptide is produced in a fed batch 30 L scale process. For this purpose, a 30 L stainless steel bioreactor (Sartorius) is equipped with all necessary sensors (DO, pH, temperature). In addition, a methanol sensor (Raven Biotech) is installed in the reactor. Fermentation is initiated from a preculture by inoculation with an optical density at 600 nm (OD 600) of 0.5. During fermentation, temperature and pH are maintained constant at 30°C and 5, respectively. The dissolved oxygen is kept at values above 30% by varying the stirrer speed, varying the gas introduction rate and adding oxygen to the feed air.The end of the batch phase is recognized by a sudden peak in the measurement value of the dissolved oxygen when the C source in the fermentation medium has been used up. In the subsequent fed phase, the methanol concentration is kept constant at 0.5% by automated addition of methanol based on the measurement value of the methanol sensor. The fermentation as described above is typically terminated after 50-60 hours.Example 4: Purification of the polypeptide of the invention from the fermentation broth:For purification, the cells are first removed by centrifugation. Coarse contaminants are then separated by filtration with a 1 μm filter before smaller particles and remaining cells are removed by filtration through a 0.45 μm membrane. The filtrate is then dialyzed against ultra pure water by tangential flow filtration using a 30 kDa membrane until a conductance remains constant. The volume is then reduced by concentration in the tangential filtration process and the concentrate is dried by lyophilization.For chromatographic purification, the dry product is dissolved in suitable running buffer and further purified either by cation exchange chromatography or IMAC. The eluate of both chromatographic methods is then dialyzed against ultra-pure water by means of tangential flow filtration and a 30 kDa membrane until a constant conductance is obtained, and then dried by lyophilization.Example 5: Biocompatibility Tests:The purified polypeptide is tested for biocompatibility by means of MTT assay. For this, L929 cells are cultured in a 96-well plate for 24 hours after sowing. Then, the recombinant polypeptide is added to the cells at concentrations of 1% and 0.1% (w / v) dissolved in cell culture medium, and the cells are cultured for a further 24 hours. For analysis, thiazolyl blue (MTT) is added to the cells at a concentration of 1 mg / ml in cell culture medium and incubated at 37°C for 2 hours. The dye solution is then removed and the formazan formed is dissolved in isopropanol. The metabolic activity of the cells is determined by measuring the absorption of the isopropanol solution at 570 nm and 650 nm. The measured values are normalized to a control group without substance addition. Cells treated with a 1% solution of the recombinant material normally exhibit a metabolic activity of at least 70% over the control group.Example 6: Functionalization of the purified productFor use in 3D printing, the purified polypeptide is functionalized by addition of methacryloyl groups. For this purpose, the purified protein is dissolved at 25° C. in a concentration of 10% (w / v) in 0.25 M CB buffer (pH 9). The pH is then adjusted to 9. At the start of the reaction, 0.1 mL methacrylic anhydride (MAA) (Sigma-Aldrich) per gram of dry protein is added to the reaction with constant stirring. Starting from the addition of MAA, direct light irradiation is avoided. The reaction is then carried out under reflux cooling for 3 hours. After 3 hours, the reaction is terminated by diluting the reaction solution with CB buffer. The reaction solution is transferred to a dialysis tube having a MWCO of 14 kDa and dialyzed against H 2 O for 3 days. The dialysis water is changed twice daily. After dialysis, the contents of the dialysis tubing are filtered through a 0.45 μm filter and lyophilized under sterile conditions. A comparative material (GelMA) is prepared analogously from gelatin (Medella Pro, Gelita).Example 7: Rheological analysis of the crosslinking propertiesFor the analysis of the crosslinking properties, the lyophilized and functionalized polypeptide is dissolved in H 2 O at a concentration of 10% (w / v) and a final concentration of 0.1% lithium phenyl 2,4,6-trimethylbenzoyl phosphinate (LAP). The crosslinking properties by free-radical reaction of the inserted methacryloyl groups (loss and storage modulus) before, during and after UV exposure (365 nm, 19.95 mW / cm 2) are measured on a rheometer (Anton pair) at 1 Hz and 0.1% deformation at 0.5 mm measurement gap and 37° C. For curing during the measurement, the material is irradiated with UV light for 180 s. GelMA is measured as a comparative material by the same method. Both materials show a rapid increase in storage modulus (G') due to the polymerization reaction upon irradiation with UV light.Example 8: Determination of functionalization by 1 H-NMRTo determine the degree of functionalization, 20 mg of unfunctionalized and 20 mg of functionalized polypeptide (from Examples 4 and 6) are dissolved in 1 ml of D 2 O and measured by means of 1 H-NMR on an Ascend 400 (Bruker) at 400 MHz. The degree of functionalization is determined by the ratios of the integral of the lysine peak in the unmodified and modified recombinant material normalized to the phenylalanine peak. The spectrum of the unmodified polypeptide (cf. FIG. 2 ) shows a higher lysine peak at about 2.9 ppm, in contrast to the modified polypeptide (cf. FIG. 3 ). In addition, the unmodified polypeptide lacks the peaks typical of methacryloyl group at about 5.3 ppm and about 5.6 ppm.Example 9: Cell Adhesion Assay with Cross-Linked Functionalized Polypeptide of the InventionFor testing cell adhesion, the functionalized polypeptide is dissolved at a concentration of 10% (w / v) in phosphate buffered saline (PBS) at a concentration of 0.1% LAP. The biotinte is placed in a 96 well ultra-low attachment (ULA) plate and fully polymerized by exposure. An 8% GelMA ink is used as a positive control, an uncoated well of the plate is used as a negative control.For colonization, primary human fibroblasts from the foreskin (FS fibroblasts) are each placed on the well with polymerized gelMA and crosslinked, inventive functionalized polypeptide (Example 7) and the uncoated plate and incubated at 37° C. for 24 hours.The cells are then examined by live dead staining with Hoechst 33342, calcein red-Oragen AM and CellTox Green and fluorescence microscopy.FS fibroblasts on the recombinant polypeptide show a lower proportion of dead cells compared to GelMA and, moreover, better cell adhesion with more uniform distribution. FS fibroblasts on the untreated plate form spheroides with a high proportion of dead cells.Example 10: 3D Bioprint of the Inventive Functionalized Polypeptide (According to Example 6)For 3D bioprinting, the functionalized polypeptide is dissolved in PBS with 0.1% LAP at 10% and 5% (w / v) concentrations as described above. The biotinte produced is then used to print a cylinder with a height of 1 mm and a radius of 1 mm on a proprietary multimaterial stereolithography (cellbricks). The material shows good printability with polymerization in the irradiated region within a few seconds and clearly defined contours. After 24 h, FS fibroblasts (20,000 cells / construct) in the construct were examined by fluorescence microscopy using live dead staining as already described in the adhesion test. FS fibroblasts show adhesion to the printed scaffold material over a period of several days with migration of cells through the sample body material.Example 11: Thermal gelling behavior of the unfunctionalized polypeptide of the invention (according to Example 4):Experience has shown that since the functionalized polypeptide according to the invention has a lower gelling point, the nonfunctionalized polypeptide according to the invention is used to measure the thermal gelling behavior. In this way it can be ensured that if the thermal gelling behavior of the unfunctionalized polypeptide is suitable, the functionalized polypeptide is also suitable. To measure the thermal gelling behavior, the nonfunctionalized polypeptide is dissolved in 10% (w / v) in H 2 O and loss and storage modulus are measured at a cooling rate of 0.5° C. / min as the temperature falls between 37° C. and 4° C. A 10% gelatin solution is used as a comparison. In contrast to gelatin, the recombinant peptide does not exhibit any thermal gelling behavior in the ambient temperature range (cf. FIG. 1 ). This can be observed by an increase in the storage modulus with a reduction in the temperature. Whereas an increase in storage modulus is already observed in the case of gelatin below about 27° C., the recombinant polypeptide shows such an increase only below about 12° C. and is therefore present in liquid form at the same concentration, in contrast to gelatin, at working temperatures typical for 3D bioprinting (room temperature about 18-22° C.).Example 13: Measurement of the circular dichroism of the unfunctionalized polypeptide of the invention (according to Example 4):To measure circular dichroism, the recombinant polypeptide is dissolved in ultra pure water at a concentration of 1 mg / ml and then filtered through a 0.22 μm syringe attachment filter. As a comparative solution, gelatin is dissolved at a concentration of 1 mg / ml and filtered through a 0.22 μm syringe attachment filter. The circular dichroism of the two solutions is measured in a J-815 CD spectrometer (Jasco) in the wavelength range 190 to 290 nm. Plots are made of the mean values of five successive measurements. The measurements were carried out for both solutions at 5° C., 20° C. and 37° C. respectively (cf. FIGS. 4 and 5 for this purpose) For gelatin, a peak typical of triple helices is found at about 220 nm at 5°C and 20°C, but not at 37°C. For the recombinant polypeptide, a positive peak typical of triple helices cannot be observed at 220 nm at any of the temperature ranges studied. This is due to the lack or lesser prolyl hydroxylation of the polypeptide of the present invention as compared to the naturally occurring polypeptide fragment.The following tables list the sequence lists (SEQ ID Nos. 1 to 8) starting with the N-terminal end and ending with the C-terminal end: Table 1: Protein sequences SEQ ID Nos. 1 and 2 Table 1: Protein sequences SEQ ID NOs: 1 and 2SEQ ID NO. 1SEQ ID NO. 2Table 2: Protein sequences SEQ ID Nos. 3 and 4Table 2: Protein sequences SEQ ID Nos. 3 and 4SEQ ID NO. 3SEQ ID NO. 4Table 3: SEQ ID NO:5: Nucleotide sequence encoding protein sequence SEQ ID NO. 1Table 3: SEQ ID NO:5: Nucleotide sequence encoding protein sequence SEQ ID NO. 1SEQ ID NO. 5Table 4: SEQ ID NO:6: Nucleotide sequence encoding protein sequence SEQ ID NO. 2Table 4: SEQ ID NO:6: Nucleotide sequence encoding protein sequence SEQ ID NO. 2SEQ ID NO. 6Table 5: SEQ ID NO:7: nucleotide sequence encoding protein sequence SEQ ID NO. 3Table 5: SEQ ID NO:7: nucleotide sequence encoding protein sequence SEQ ID NO. 3SEQ ID NO. 7Table 6: SEQ ID NO:8: nucleotide sequence encoding protein sequence SEQ ID NO. 4Table 6: SEQ ID NO:8: nucleotide sequence encoding protein sequence SEQ ID NO. 4SEQ ID NO. 8References included in the specificationThis list of documents cited by the applicant has been produced in an automated manner and is only included for the better information of the reader. The list is not part of the German patent application or utility model application. The DPMA does not take any adhesion for any faults or omissions.Patent Literature citedEP 3 018 531 B1

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Claims

A method for producing an ink for 3D printing, wherein a polypeptide is put into a solvent, characterized in that the polypeptide is a human collagen-based polypeptide having a proportion of hydroxypropylproline of 5% at maximum, based on the total number of amino acids of the polypeptide.The method of claim 1, wherein the polypeptide has a level of hydroxyquinoline of at most 4%, preferably at most 3%, based on the total number of amino acids of the polypeptide.The method of claim 1 or 2, wherein the polypeptide is functionalized with at least one crosslinkable group.The method according to any one of claims 1 to 3, wherein the crosslinkable group is a photocrosslinkable group.The method of any one of claims 1 to 4, wherein the polypeptide is one that is in the liquid state in an aqueous solution at concentrations of 10 weight percent per volume (% w / v) at temperatures of 18°C and greater.The method of any one of claims 1 to 5, wherein the polypeptide has a molecular weight in the range of 20 to 200 daltons.The method of any one of claims 1 to 6, wherein the ink contains biological cells.Ink for 3D printing with a polypeptide based on human collagen, which has a proportion of hydroxypropylproline of at most 5%, based on the total number of amino acids of the polypeptide.The ink according to claim 8, obtainable by a process according to any one of claims 1 to 7.The ink according to claim 8 or 9 for use in medicine.A method for manufacturing a 3D scaffold using an ink according to any one of claims 8 to 10, wherein the polypeptide is cured by a 3D printing method.The method of claim 11, wherein the polypeptide is functionalized with at least one crosslinkable group.Method according to claim 11 or 12, wherein light-based shaping, preferably by light-induced chemical reaction, is used in the 3D printing method.The method of any one of claims 11 to 13, wherein the ink contains biological cells.3D framework comprising cross-linked polypeptides, wherein the polypeptides are polypeptides based on human collagen, each having a proportion of hydroxyquinoline of at most 5%, based on the total number of amino acids of the respective polypeptide.The 3D framework of claim 15, additionally comprising biological cells.3D framework according to Claim 15 or 16, obtainable by a process according to one of Claims 11 to 14.3D-scaffolding according to any one of claims 15 to 17 for use in medicine.

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