Novel bioartificial vascular grafts and methods for their fabrication

Abstract

The present invention relates to a novel bio-ink for preparing bioartificial vascular grafts which are highly useful for vascular and cardiovascular research and therapy, and in particular for surgical vascular and cardiovascular repair and bypass surgery. The bio-ink composition of the invention comprises a peptide-grafted alginate and smooth muscle cells, endothelial cells or cells which are capable of developing into endothelial cells. According to the invention, the peptides grafted onto the alginate are derived from an extracellular matrix protein or mimic such proteins. The invention also relates to a method for manufacturing a bioartificial vascular graft comprising the printing of the bio-ink composition of the invention. Bioartificial vascular grafts, which are obtainable by the method of the invention, are also encompassed by the invention. In yet another aspect, the invention relates to the use of the bio-ink for in vitro drug screening and as an in-vitro training model for endovascular or surgical training.

Description

[0001]NOVEL BIOARTIFICIAL VASCULAR GRAFTS AND METHODSFOR THEIR FABRICATIONThe present invention relates to a novel bio-ink for preparing bioartificial vascular grafts whichare highly useful for vascular and cardiovascular research and therapy, and in particular forsurgical vascular and cardiovascular repair and bypass surgery. The bio-ink composition ofthe invention comprises a peptide-grafted alginate and smooth muscle cells, endothelial cellsor cells which are capable of developing into endothelial cells. According to the invention, thepeptides grafted onto the alginate are derived from an extracellular matrix protein or mimicsuch proteins. The invention also relates to a method for manufacturing a bioartificial vasculargraft comprising the printing of the bio-ink composition of the invention. Bioartificial vasculargrafts, which are obtainable by the method of the invention, are also encompassed by theinvention. In yet another aspect, the invention relates to the use of the bio-ink for in vitro drugscreening. TECHNICAL BACKGROUNDCoronary or peripheral bypass surgery due to arteriosclerotic stenosis or occlusion remainsone of the major medical procedures of today (Kullo et al., 2016; Head et al., 2017). Addition-ally, an increasing number of civilian and military vessel traumas have been reported duringthe previous five decades associated with an increase of vascular repair (Caps, 1998; Haneyet al., 2020). In the majority of cases, autologous vein or less often arterial vessels are im-planted as bypass grafts or vascular conduits.Due to the nature of autologous approaches, the number of grafts is limited which could bedecisive to treatment in case of re-operation or co-morbidities of the vascular system (Calis-kan et al., 2020; Gallo et al., 2022; Xenogiannis et al., 2021). Artificial polymer grafts are stillassociated with poor patency rates and high infection risk or are not suitable in various treat-ments especially for small vessel grafting e.g., coronary bypass surgery (Twine et al., 2010).Accordingly, the ideal and rapidly available vascular graft remains a challenge of modernmedicine - not only for the surgical team but also for biomedical research and health caresystems.Recent developments in tissue engineering in general and especially in 3D bioprinting haveraised the legitimate hope to fabricate a bioartificial vascular graft for cardiovascular surgery(Sasmal et al., 2018; Matai et al. 2020). A number of preliminary studies have describedbioprinting strategies for vessel-like structures ranging from customized to commercial, fromco-axial to laser-based bioprinting and presenting a wide range of different bio-inks (Matai etal., 2020). For example, Zhang and co-workers described a bioprinted perfusable vasculatureconduit fabricated from human umbilical vein smooth muscle cells (SMCs) encapsulated insodium alginate (Zhang et al., 2015). Hong and colleagues demonstrated a vascular constructusing a gelatin–tyramine bio-ink (Hong et al., 2019). A similar approach was described by Cuiand co-workers demonstrating a small-diameter vasculature with smooth muscle and endo-thelium (Cui et al., 2019).However, the majority of these approaches did not provide the necessary bioartificial integrity,a pipeline for clinical translation and were mainly focused on in-vitro disease modelling (Sas-mal et al., 2018). Bioartificial vascular grafts for surgical implantation are mostly bioengi-neered from components of the extracellular matrix, are acellular and therefore lack the func-tional biology of human vessels (Dahl et al., 2011). In addition, limited cell sources have beendescribed as a major problem of these tissue-engineering approaches (Ott et al., 2011). Con-sequently, none of these former studies has yet provided a bioartificial vascular graft combin-ing the required biomimetic characteristics of human vessels and a strategy for clinical trans-lation.As a consequence, there is an unfulfilled demand in human-scale bioartificial vascular graftswhich can be used in cardiovascular therapy and exert biomechanics and biomimetics thatresemble human vessels, a high capability of biointegration, and low thrombogenicity. Thebioartificial vascular grafts should be available by reproducible, scalable and reliable GMPprocesses. Moreover, the manufacturing process should be short and scalable to allow amore “bedside” orientated approach.The present invention has overcome the problems experienced in the past and provides ad-ditional advantages as well. The present invention for the first time demonstrates that bioar-tificial vascular grafts having human-scale structure and functional integrity can be obtainedby 3D bioprinting. The present invention provides a novel bio-ink which comprises an alginate,such as sodium alginate (SA) and one or more peptides, such as peptides derived from pro-teins of the extracellular matrix (EM). Endothelial or SMCs are integrated into the vessel wallas part of the printing process to create a vein-like vascular graft ready for clinical application.The grafts of the present invention exert biomechanical properties and characteristics thatrender them suitable for being used in clinical settings for repairing or replacing dysfunctionalor diseased vessels.SUMMARY OF THE INVENTIONAccordingly, in a first aspect the present invention relates to a bio-ink composition which canbe used for bioprinting a vascular graft, said bio-ink composition comprising:(a) a peptide-grafted alginate; and(b) SMCs, endothelial cells or cells which are capable of developing into endothelialcells.The bio-ink composition according to the first aspect of the invention is suitable for beingprinted to a 3D bioartificial vascular graft by use of a 3D printer. Therefore, in a second aspect,the present invention relates to a method for manufacturing a bioartificial vascular graft, com-prising (a) loading the bio-ink composition according to the first aspect of the invention into a3D printer which is capable of printing viscous hydrogels;(b) contacting the bio-ink composition with a crosslinking solution comprising a divalentand / or trivalent cation in the 3D printer to induce crosslinking of the alginate;(c) printing the bio-ink to obtain the bioartificial vascular graft.In a third aspect, the present invention relates to a bioartificial vascular graft obtainable bythe method according to the second aspect of the present invention. The bioartificial vasculargraft according to the third aspect of the invention can be used as a vascular conduit or by-pass material.In a fourth aspect, the present invention relates to the bioartificial vascular graft according tothe third aspect of the invention for use in medicine, and in particular for use in vascular orcardiovascular therapy, such as bypass surgery.In a fifth aspect, the present invention relates to the use of a bio-ink composition accordingto the first aspect of the invention for manufacturing a bioartificial vascular graft, such asvascular conduit and bypass material. The bioartificial vascular graft can be used as a vas-cular conduit or bypass material.In a sixth aspect, the present invention relates to the use of a bio-ink composition accordingto the first aspect of the invention for in vitro drug screenings or an in-vitro training model forendovascular or surgical training.DETAILED DESCRIPTIONThe bio-ink composition of the present invention provides a 3D matrix for printing a bioartificialvessel that can be applied for research purposes, for in vitro disease modelling or in medicaltherapy. The matrix integrates high tensile strength, elasticity and a favorable environmentfor cell engraftment which are essential requirements for mimicking the characteristics of nat-ural vessels (Matai et al., 2020). Accordingly, the bio-ink composition of the present inventionis highly suitable for manufacturing of vascular conduits and bypass material.The bio-ink of the present invention comprises, as a first component, a peptide-grafted algi-nate, i.e. an alginate to which one or more peptides have been covalently coupled. As usedherein, the term “alginate” refers to a salt of alginic acid. Alginic acid is a polysaccharideconsisting of a linear copolymer with homopolymeric blocks of (1-4)-linked β-D-mannuronate(M) and α-L-guluronate (G) residues, respectively, covalently linked together in different se-quences or blocks. The monomers may appear in homopolymeric blocks of consecutive G-residues (G-blocks), consecutive M-residues (M-blocks) or alternating M and G-residues(MG-blocks). Alginic acid naturally occurs in brown algae. It is a hydrophilic molecule which,upon hydration or interaction with divalent cations, forms a hydrogel. Metal salts of alginicacid, such as sodium alginate and calcium alginate, are in wide use for different purposes,for example as thickening agents in foods and cosmetics, as an ingredient in various phar-maceutical products, and as a biomaterial for tissue regeneration. Alginates have also beencontemplated for bioprinting (Fan et al., 2013; Hu et al., 2021). Although sodium alginateexerts some beneficial physicochemical properties, such as biocompatibility, non-toxicity andnon-immunogenicity, its capability of cell engraftment is limited (Fan et al., 2013; Matai et al.,2020; Hu et al., 2021). In addition, the biomechanical properties of sodium alginate are ratherlimited. The present invention overcomes this problem by modifying the alginate with EMprotein fragments. The M / G ratio of the alginate that is used in the methods and compositionsof the invention can vary between 10:1 and 1:10, and will preferably be about 10:1, 9:1, 8:1,7:1, 6:1, 5:1, 4:1, 3:1, 2:1, and 1:1. In a preferred embodiment, the M / G ratio of the alginatethat is used in the methods and compositions of the invention is about 1:1. It is also possibleaccording to the invention to use an alginate that completely consists of a mannuronate, suchas a (1-4)-linked β-D-mannuronate.In a preferred embodiment, the alginate which is present in the bio-ink composition of theinvention is sodium alginate. In another preferred embodiment, the alginate which is presentin the bio-ink composition of the invention is calcium alginate. In yet another preferred em-bodiment, the alginate which is present in the bio-ink composition of the invention is a mixtureof sodium alginate and calcium alginate.The present invention is based, amongst others, on the insight that the positive effects ofsodium alginate and EM proteins can be combined by grafting smaller peptides, e.g. peptidesobtained by hydrolysis of EM proteins or artificial peptides that comprise an amino acid se-quence which occurs in EM proteins, in particular collagen, onto sodium alginate to providea grafted matrix that can be used as a bio-ink. In the bio-ink composition of the invention, thealginate is present in the form of peptide-alginate complexes in which the alginate is cova-lently coupled to at least one peptide, i.e., one defined type of peptide, such as a peptidederived from the hydrolysis of collagen or manufactured by peptide synthesis.According to the invention, the peptide which is coupled to the alginate preferably comprisesan amino acid sequence which is derived from an EM protein, such as a human EM protein.As used herein, “derived from” means that the amino acid sequence in the peptide occurs inidentical or highly similar form in the amino acid sequence of a native EM protein. Accordingly,the peptide may comprise or consist of an amino acid sequence which is present in a naturallyoccurring. EM protein. Alternatively, the peptide may comprise or consist of an amino acidsequence which resembles an amino acid sequence which is present in a naturally occurringEM protein. The EM protein preferably is a human EM protein. Also encompassed by theterm “derived from an EM protein” or “EM protein-derived” are peptides which structurallymimic an EM protein, preferably a human EM protein. Such peptides include peptides thatare recognized by a protease that specifically recognizes the corresponding EM protein. Forexample, the synthetic peptide N-[3-(2-Furyl)acryloyl]-L-leucyl-glycyl-L-prolyl-L-alanine(FALGPA) which was used in the below examples resembles a partial structure of the humancollagen and is hence recognized by a collagenase enzyme. In a preferred embodiment, theEM protein-derived peptide is recognized by the collagenase of Clostridium histolyticum, anenzyme that is widely commercialized in kits for measuring collagenase activity.The EM protein from which the one or more peptides can be derived are preferably selectedfrom the group consisting of collagen, tropocollagen, elastin, tropoelastin, fibrillin I, fibrillin II,fibrillin III, fibrillin IV, fibronectin vitronectin and laminin. In one embodiment, the peptide usedin the bio-ink composition of the invention comprises a peptide which is derived from a colla-gen protein. The collagen protein can be a collagen I protein, II protein, a collagen III protein,a collagen IV protein, a collagen V protein, a collagen VI protein, a collagen VII protein, acollagen VIII protein, a collagen IX protein, a collagen X protein, a collagen XI protein, acollagen XII protein, a collagen XIII protein, a collagen XIV protein, a collagen XV protein, acollagen XVI protein, a collagen XVII protein, a collagen XVIII protein, a collagen XIX protein,a collagen XX protein, a collagen XXI protein, a collagen XXII protein, a collagen XXIII protein,a collagen XXIV protein, a collagen XXV protein, a collagen XXVI protein, a collagen XXVII,a collagen XXVIII, or a collagen XIX. It is particular preferred that the peptide is derived froma collagen I or collagen II protein, and more preferably from the human collagen I or collagenII protein. In another embodiment, the peptide used in the bio-ink composition is derived froma proteoglycan, preferably, from an aggrecan, decorin, or perlecan.In a particularly preferred embodiment, the peptide in the peptide-grafted alginate is derivedfrom collagen, i.e., the peptide comprises or consists of an amino acid sequence that occursin collagen, preferably human collagen, or is highly similar to an amino acid sequence thatoccurs in collagen, or mimics a collagen molecule.In another embodiment, the alginate is covalently coupled to a combination of different pep-tides, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides, such as EM protein-derived peptides.For example, the alginate may be covalently coupled to a first EM-derived peptide and asecond EM-derived peptide wherein both peptides differ from each other in terms of theiramino acid sequence. Thus, the invention also relates to a bio-ink composition which com-prises a peptide-grafted alginate, in which the alginate is grafted with 2 or more differentpeptides.In a preferred embodiment, the alginate is present in the form of peptide-alginate complexesin which the alginate is covalently coupled to a first collagen-derived peptide and a secondcollagen-derived peptide which differs from the first peptide in terms of its amino acid se-quence. For example, in one embodiment, the first collagen-derived peptide is derived fromcollagen I, and the second collagen-derived peptide is derived from collagen II. In anotherembodiment, the first collagen-derived peptide is derived from collagen I, and the secondcollagen-derived peptide is derived from collagen III. In yet another embodiment, the firstcollagen-derived peptide is derived from collagen I, and the second collagen-derived peptideis derived from collagen IV. In yet another embodiment, the first collagen-derived peptide isderived from collagen I, and the second collagen-derived peptide is derived from collagen V.In yet another embodiment, the first collagen-derived peptide is derived from collagen I, andthe second collagen-derived peptide is derived from collagen VI. In yet another embodiment,the first collagen-derived peptide is derived from collagen I, and the second collagen-derivedpeptide is derived from collagen VII. In yet another embodiment, the first collagen-derivedpeptide is derived from collagen I, and the second collagen-derived peptide is derived fromcollagen VIII. In yet another embodiment, the first collagen-derived peptide is derived fromcollagen I, and the second collagen-derived peptide is derived from collagen IX. In yet anotherembodiment, the first collagen-derived peptide is derived from collagen I, and the secondcollagen-derived peptide is derived from collagen X.Alternatively, the alginate may also be covalently coupled to a combination of a first collagen-derived peptide and a second peptide which is derived from another EM protein, i.e., an EMprotein other than collagen. The second EM-derived peptide can be derived from any of theEM proteins referred to above. In one embodiment, the second peptide is derived from theEM protein laminin. In another embodiment, the second peptide is derived from the EM pro-tein elastin. In still another embodiment, the second peptide is derived from the EM proteintropoelastin. In another embodiment, the second peptide is derived from the EM protein fi-brillin I, fibrillin II, fibrillin III, or fibrillin IV. In still another embodiment, the second peptide isderived from the EM protein fibronectin or vitronectin.Each of the one or more peptides which are covalently coupled to the alginate preferably hasa size of 3-50 amino acids. In one embodiment, each of the peptides has a size of 3-45 aminoacids, 3-40 amino acids, 3-35 amino acids, 3-30 amino acids, 3-25 amino acids, 3-20 aminoacids, 3-15 amino acids, 3-10 amino acids, 3-9 amino acids, 3-8 amino acids, 3-7 aminoacids, 3-6 amino acids, 3-5 amino acids, or 3-4 amino acids. In another embodiment, each ofthe peptides has a size of 4-45 amino acids, 4-40 amino acids, 4-35 amino acids, 4-30 aminoacids, 4-25 amino acids, 4-20 amino acids, 4-15 amino acids, 4-10 amino acids, 4-9 aminoacids, 4-8 amino acids, 4-7 amino acids, 4-6 amino acids, or 4-5 amino acids. Preferably,each of the peptides has a size of 3 amino acids, 4 amino acids, 5 amino acids, 6 aminoacids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids. A size of 4-8 or 4-10amino acids is particularly preferred. It is particular preferred that the above sizes refer to apeptide which is derived from a collagen protein, more preferably a collagen I or collagen IIprotein, such as the human collagen I or collagen II protein.In one preferred embodiment, the peptide in the bio-ink composition of the present inventionis derived from a collagen I protein and comprises or consists of the tetramer sequence NH2-L-G-P-A-COOH (SEQ ID NO:1). In another preferred embodiment, the peptide in the bio-inkcomposition is derived from a collagen I protein and comprises or consists of the sequenceN-(3-[2-Furyl]acryloyl)-L-G-P-A (SEQ ID NO:2). In yet another preferred embodiment, thepeptide in the bio-ink composition is derived from a collagen I protein and comprises or con-sists of the sequence NH2-D-G-E-A-COOH (SEQ ID NO:9). In yet another preferred embod-iment, the peptide in the bio-ink composition is derived from a collagen I protein and com-prises or consists of the sequence NH2-G-F-O-G-E-R-COOH (SEQ ID NO:10), wherein O ishydroxyproline. In another preferred embodiment, the peptide in the bio-ink composition isderived from laminin and comprises or consists of the hexamer sequence NH2-S-I-K-V-A-V-COOH (SEQ ID NO:3). In yet another preferred embodiment, the peptide in the bio-ink com-position is derived from elastin and comprises or consists of the hexamer sequence NH2-V-G-V-A-P-G-COOH (SEQ ID NO:4). In yet another preferred embodiment, the peptide in thebio-ink composition is derived from laminin and comprises or consists of the hexamer se-quence NH2-V-G-V-A-P-G-COOH (SEQ ID NO:5). In yet another preferred embodiment, thepeptide in the bio-ink composition comprises or consists of the NH2-R-G-D-COOH. In yetanother preferred embodiment, the peptide in the bio-ink composition comprises or consistsof the sequence NH2-K-Q-A-G-D-V-COOH (SEQ ID NO:6). Such peptides are discussed inmore detail in Hashimoto et al. 2004. Without wishing to be bound by theory, it is assumedthat EPCs, SMCs, and endothelial cells recognize these sequences such that their integrationinto the alginate matrix is promoted.It is of course also possible to produce, either synthetically or by recombinant expression,hybrid peptides which encompass two or more amino acid sequences which are derived fromdifferent EM proteins. For example, hybrid peptides can be produced which combine peptidesequences derived from laminin and elastin. Such peptides include, e.g., NH2-K-S-I-R-V-A-V-A-P-G-COOH (SEQ ID NO:7) and NH2-K-S-I-R-V-G-V-G-P-G-COOH (SEQ ID NO:8). Theskilled person will be readily able to synthesize or recombinantly express hybrid peptideswhich combine sequences from two or more EM proteins.According to the invention, it is preferred that the alginate is present in the form of peptide-alginate complexes in which the alginate is covalently coupled to one or more peptides thatcomprise or consist of the amino acid sequence set forth in SEQ ID NOs:1-8 or a variant ofany of these. As used herein, an amino acid sequence is considered a "variant" if it differsfrom one of the amino acid sequences set forth in SEQ ID NOs:1-8 by no more than 2 aminoacid exchanges, and more preferably by no more than 1 amino acid exchange. Thus, it ispreferred in one embodiment that the alginate is coupled to a peptide that comprises or con-sists of the sequence of SEQ ID NO:1 or a sequence that differs from the sequence of SEQID NO:1 by 1 or 2 amino acid exchanges. In another embodiment the alginate is coupled toa peptide that comprises or consists of the sequence of SEQ ID NO:2 or a sequence thatdiffers from the sequence of SEQ ID NO:2 by 1 or 2 amino acid exchanges. In yet anotherembodiment the alginate is coupled to a peptide that comprises or consists of the sequenceof SEQ ID NO:3 or a sequence that differs from the sequence of SEQ ID NO:3 by 1 or 2amino acid exchanges. In another embodiment the alginate is coupled to a peptide that com-prises or consists of the sequence of SEQ ID NO:4 or a sequence that differs from the se-quence of SEQ ID NO:4 by 1 or 2 amino acid exchanges. In another embodiment the alginateis coupled to a peptide that comprises or consists of the sequence of SEQ ID NO:5 or asequence that differs from the sequence of SEQ ID NO:5 by 1 or 2 amino acid exchanges. Inanother embodiment the alginate is coupled to a peptide that comprises or consists of thesequence of SEQ ID NO:6 or a sequence that differs from the sequence of SEQ ID NO:6 by1 or 2 amino acid exchanges. In another embodiment the alginate is coupled to a peptide thatcomprises or consists of the sequence of SEQ ID NO:7 or a sequence that differs from thesequence of SEQ ID NO:7 by 1 or 2 amino acid exchanges. In another embodiment the algi-nate is coupled to a peptide that comprises or consists of the sequence of SEQ ID NO:8 or asequence that differs from the sequence of SEQ ID NO:8 by 1 or 2 amino acid exchanges. Inyet another embodiment the alginate is coupled to a peptide that comprises or consists of thesequence of SEQ ID NO:9 (Mehta et al.2015; Phipps, et al.2010) or a sequence that differsfrom the sequence of SEQ ID NO:9 by 1 or 2 amino acid exchanges. In still another embodi-ment the alginate is coupled to a peptide that comprises or consists of the sequence of SEQID NO:10 (Zhang et al.2003; Castillo-Briceño at al.2011; Liao et al.2024) or a sequence thatdiffers from the sequence of SEQ ID NO:10 by 1 or 2 amino acid exchanges.It is particularly preferred according to the invention that the alginate is present in the form ofpeptide-alginate complexes in which the alginate is covalently coupled to one or more pep-tides that comprise or consist of the amino acid sequence set forth in SEQ ID NOs:1, SEQ IDNOs:2, SEQ ID NOs:9, or SEQ ID NOs:10.Accordingly, in a preferred aspect, the invention relates to a bio-ink composition suitable forbeing printed into a bioartificial vascular graft, said composition comprising:(a) a peptide-grafted alginate, wherein said peptide comprises or consists of a collagen-derived sequence, preferably the amino acid sequence set forth in SEQ ID NOs:1,SEQ ID NOs:2, SEQ ID NOs:9, or SEQ ID NOs:10; and(b) smooth muscle cells, endothelial cells or cells which are capable of developing intoendothelial cells.The one or more EM-derived peptides can be coupled to the alginate, preferably sodium orcalcium alginate, by different chemical methods for linking peptides to polysaccharides. Forexample, the peptide or peptides can be covalently coupled to the alginate via an amidelinkage in the presence of 1-ethyl-(dimethylaminopropyl) carbodiimide and N-hydroxy sul-fosuccinimide. As described in the below examples, the carbodiimide reaction was used tomediate the crosslinking between sodium alginate and an EM-derived peptide, the FALGPApeptide (N-(3-[2-Furyl]acryloyl)-L-G-P-A SIGMA-Aldrich). 0.35 g sodium alginate was dis-solved in 0.2 M MES (2-(N-morpholino) ethanesulfonic acid) buffer containing 0.3 M NaCl,pH 5.5. Then, 0.035 g N-hydroxy sulfosuccinimide (NHS) and 0.035 g N-(3-Dimethyla-minopropyl)-N-ethylcarbodiimide hydrochloride (EDC) were added step by step into the solu-tion at room temperature with magnetic stirring for 20 min. The pH was slowly increased to 7and then peptides were added into the solution facilitating the grafting for 8 h. The 1% alginatederivative solution so produced was purified by dialysis through a 6-8 KD molecular weightcut-off dialysis tube for three days. The dialyzed product was finally freeze-dried with LyoVacGT2 to obtain a purified alginate derivative powder. The dried samples were stored at -20°C.The overall amount of peptide which is coupled to the alginate in the peptide-alginate com-plexes can be between 1% and 25%, based on the overall weight of β-D-mannuronate in thealginate (w / w). More preferably, the amount of peptide in the peptide-alginate complexes isbetween 1% and 20% (w / w), between 1% and 15% (w / w), between 1% and 12% (w / w), orbetween 1% and 10% (w / w), based on the overall weight of β-D-mannuronate in the alginate.More preferably, the amount of peptide in the peptide-alginate complexes is between 2% and25% (w / w), between 2% and 20% (w / w), between 2% and 15% (w / w), between 2% and 12%(w / w), or between 2% and 10% (w / w), based on the overall weight of β-D-mannuronate in thealginate. More preferably, the amount of peptide in the peptide-alginate complexes is be-tween 3% and 25% (w / w), between 3% and 20% (w / w), between 3% and 15% (w / w), between3% and 12% (w / w), or between 3% and 10% (w / w), based on the overall weight of β-D-mannuronate in the alginate. More preferably, the amount of peptide in the peptide-alginatecomplexes is between 4% and 25% (w / w), between 4% and 20% (w / w), between 4% and15% (w / w), between 4% and 12% (w / w), or between 4% and 10% (w / w), based on the overallweight of β-D-mannuronate in the alginate.Stated differently, the overall amount of peptide which is coupled to the alginate in the pep-tide-alginate complexes can be between 0.5% and 12.5% (w / w), based on the overall weightof the alginate. More preferably, the amount of peptide in the peptide-alginate complexes isbetween 0.5% and 10% (w / w), between 0.5% and 7.5% (w / w), between 0.5% and 6% (w / w),or between 0.5% and 5% (w / w), based on the overall weight of the alginate. More preferably,the amount of peptide in the peptide-alginate complexes is between 1% and 12.5% (w / w),between 1% and 10% (w / w), between 1% and 7.5% (w / w), between 1% and 6% (w / w), orbetween 1% and 5% (w / w), based on the overall weight of the alginate. More preferably, theamount of peptide in the peptide-alginate complexes is between 1.5% and 12.5% (w / w), be-tween 1.5% and 10% (w / w), between 1.5% and 7.5% (w / w), between 1.5% and 6% (w / w), orbetween 1.5% and 5% (w / w), based on the overall weight of the alginate. More preferably,the amount of peptide in the peptide-alginate complexes is between 2% and 12.5% (w / w),between 2% and 10% (w / w), between 2% and 7.5% (w / w), between 2% and 6% (w / w), orbetween 2% and 5% (w / w), based on the overall weight of the alginate.The presence and percentage of coupled peptides in the alginate can be confirmed by differ-ences in the absorbances compared to an unmodified alginate control, as further describedin the below Example 1. Alternatively, the presence and percentage of coupled peptides inthe alginate can be confirmed by mass spectroscopy, as described in Fan et al.2013.The bio-ink composition of the invention comprises, as a second component, SMCs, endo-thelial cells or cells which are capable of developing into endothelial cells. The cells are pref-erably autologous in relation to the subject that shall receive the bioartificial vascular graft,i.e., the cells have been derived from the subject that shall receive the printed graft. However,allogenic cells may also be used, i.e., cells obtained from a subject of the same species notbeing the patient to be treated. For example, hypoimmunogenic allogenic endothelial cellscan be used in order to avoid a strong immunogenic reaction in the patient receiving the graft.In one embodiment, the cells that are added to the bio-ink composition of the invention areendothelial cells. As used herein, the term “endothelial cells” generally refers to cells that linethe interior surface of blood vessels or lymphatic vessels. Preferably, the endothelial cellspresent in the bio-ink of the invention are vascular endothelial cells, i.e., cells that form theinner cellular lining of blood vessels, including arteries, veins and capillaries. Endothelial cellsexpress the cells surface markers CD31, CD34 and ICAM1 and can thus be easily identified,e.g., by flow cytometry, and in particular cell fluorescence-activated cell sorting (FACS). Pref-erably, the endothelial cells do not express CD45.Limited cell sources have been one of the major factors for failure of tissue-engineering ap-proaches in the past (Ott et al., 2011). The present invention deliberately omits muscle cellsfrom the Tunica media and provides a vein-like graft preferably consisting solely of endothelialcells and connective tissue. The endothelial cells used in the bio-inks of the invention can bederived from different sources. For example, the endothelial cells can be Human UmbilicalVein Endothelial Cells (HUVEC), mononuclear-derived endothelial-like cells, endothelial cellsderived from induced pluripotent stem cells (iPS) and blood-derived endothelial cells. In apreferred embodiment, the endothelial cells are HUVEC. In another preferred embodiment,the endothelial cells are derived from mononuclear-derived endothelial-like cells. In yet an-other embodiment, the endothelial cells are derived from iPS. Preferably, the endothelial cellsused in the bio-inks of the invention are human cells. In yet another embodiment, the endo-thelial cells are derived from punch biopsies.Apart from or in addition to the differentiated endothelial cells, the bio-inks of the inventionmay also comprise cells which are capable of developing into endothelial cells. A preferredexample of cells having the capacity to develop into endothelial cells are endothelial progen-itor cells (EPCs). As used herein, the term “EPCs” refers to immature cells that have thecapacity to differentiate into mature endothelial cells and express the cell surface markersCD31, CD34, CD146, CD309, while at the same time they do not express CD45 and CD14(Mead et al., 2008; Emontzpohl et al., 2017). EPCs can be identified by morphological anal-ysis and / or flow cytometry, and in particular FACS.EPCs for use in the bio-ink of the invention have been extensively described in the prior artand can be readily obtained from whole blood samples as described in the literature before(Baudin et al. 2007, Mead et al., 2008). Briefly, mononuclear cells are isolated from bloodusing Ficoll density gradient centrifugation according to standard protocols, followed by wash-ing with phosphate-buffered saline (PBS). Cells are cultured using endothelial basal medium2 (EBM-2; Lonza, Basel, Switzerland) supplemented with EBM-2-MV-SingleQuots (Lonza,Basel, Switzerland). Cells in suspension are removed after 3 days of culture and adherentEPCs are passaged at 80–90% confluence (Mead et al., 2008; Emontzpohl et al., 2017; Konget al., 2021). All cell cultivation steps are performed in a humidified atmosphere at 37°C and5% CO2. Apart from this manual procedure, EPCs can also be obtained via automated cellprocessing e.g., by using the CliniMACS Prodigy (Miltenyi) in combination with magnetic beadsorting.SMCs can also be used in the bio-inks of the invention. These cells are regularly used intissue engineering for the construction of vascular grafts. Preferably, the SMCs used in thebio-inks of the invention are of the contractile type which means that they are spindle-shapedcells which provide contractile filaments. SMCs can be obtained by differentiating autologousprecursor cells from samples or by punch biopsy as described in Simper et al.2002 or Kolsteret al. 2017).The number of cells that are added to the bio-ink composition of the invention may vary de-pendent on the graft to be produced. Typically, the bio-ink composition will comprise a totalof 1x103 to 1x108 cells per ml, and preferably 1x105 to 1x107 cells per ml, and more preferably1x106 to 5x107 cells per ml. In one embodiment, the bio-ink composition will comprise 1x103to 1x108 endothelial cells per ml, and preferably 1x105 to 1x107 endothelial cells per ml, andmore preferably 1x106 to 5x107 endothelial cells per ml. In another embodiment, the bio-inkcomposition will comprise 1x103 to 1x108 EPCs, and preferably 1x105 to 1x107 EPCs, andmore preferably 1x106 to 5x107 EPCs. In yet another embodiment, the total number of endo-thelial cells and EPCs which are present in the bio-ink composition of the invention will be1x103 to 1x108 cells, and preferably 1x105 to 1x107 cells, and more preferably 1x106 to 5x107cells. In another embodiment, the bio-ink composition will comprise 1x103 to 1x108 SMCs,and preferably 1x105 to 1x107 SMCs, and more preferably 1x106 to 5x107 SMCs. Accordingto the invention, the SMCs, endothelial cells and / or EPCs are mixed into the bio-ink compo-sition of the invention. When printing the bio-ink composition, the cells become embeddedinto the graft, thereby providing stable biomechanical characteristics to the graft which distin-guish the grafts of the invention from other preliminary approaches.The bio-ink composition of the invention can be prepared by mixing the peptide-alginate com-plexes with an appropriate number of SMCs, endothelial cells and / or EPCs. Optionally, fur-ther excipient compounds may be added, e.g., compounds that are used to modulate thefluidity of the bio-ink to adapt it to a specific printing device. The aqueous bio-ink compositioncan then be cross-linked by the addition divalent and trivalent cations. For example, the bio-ink composition can be cross-linked by the addition Ca2+, Sr2+, Ba2+, Cu2+, Zn2+, Mn2+, Fe2+,Cr3+, or Fe3+ cations. It is preferred herein to effect cross-linking by Ca2+ cations. The cross-linking can be achieved during the printing process, preferably by mixing the aqueous bio-inkcomposition during the printing process with a solution containing the cation, e.g., a CaCl2solution. The mixing of the bio-ink composition can be carried out in the print head of the 3Dprinter.Also provided is a method for manufacturing a bioartificial vascular graft in which the bio-inkcomposition of the invention is printed to a 3D bioartificial vascular graft by use of a 3D printer.The method comprises the following steps:(a) loading a bio-ink composition according to the first aspect of the invention into a 3Dprinter which is capable of printing viscous hydrogels;(b) contacting the bio-ink composition with a crosslinking solution comprising a divalentand / or trivalent cation in the 3D printer to induce crosslinking of the alginate;(c) printing the bio-ink to obtain the bioartificial vascular graft.In the first step of the method, a bio-ink composition as described herein above is loaded intoa 3D printer which is capable of printing viscous hydrogels. The bio-ink composition prefera-bly is an aqueous composition. For this purpose, a solution of an alginate is provided to whichEM protein-derived peptides have been coupled. The amount of the alginate (including thepeptide-grafted alginate) in the bio-ink composition can range from 1.0% to about 25.0%(w / w), based on the overall weight of the aqueous bio-ink composition. An amount of 5.0%(w / w) based on the overall weight of the aqueous bio-ink composition is particularly preferred.Preferably, the amount of the alginate (including the peptide-grafted alginate) in the aqueousbio-ink composition will be about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%,about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 11.0%, about 12.0%, about 13.0%,about 14.0%, about 15.0% (w / w), about 16.0% (w / w), about 17.0% (w / w), about 18.0% (w / w),about 19.0% (w / w), about 20.0% (w / w), about 21.0% (w / w), about 22.0% (w / w), about 23.0%(w / w), about 24.0% (w / w), or about 25.0% (w / w), based on the overall weight of the aqueousbio-ink composition. In a particularly preferred embodiment, the amount of the alginate (pep-tide-grafted alginate) in the aqueous bio-ink composition is between 2.0 to 5.0%, and morepreferably 4.0 to 5.0%, based on the overall weight of the aqueous bio-ink composition.Then, the SMCs, endothelial cells and / or EPCs are added to the aqueous alginate solution.The cells are added in the numbers set forth above, i.e., a total of 1x103 to 1x108 cells per mlof the aqueous alginate solution, preferably 1x106 to 5x107.In one embodiment, the amount of the alginate (including the peptide-grafted alginate) in theaqueous bio-ink composition is 2.0%, based on the overall weight of the aqueous bio-inkcomposition, and a total of 1x103 to 1x108 cells per ml of the aqueous alginate solution. Inanother embodiment, the amount of the alginate (including the peptide-grafted alginate) inthe aqueous bio-ink composition is 3.0%, based on the overall weight of the aqueous bio-inkcomposition, and a total of 1x103 to 1x108 cells per ml of the aqueous alginate solution. In yetanother embodiment, the amount of the alginate (including the peptide-grafted alginate) inthe aqueous bio-ink composition is 4.0%, based on the overall weight of the aqueous bio-inkcomposition, and a total of 1x103 to 1x108 cells per ml of the aqueous alginate solution. In yetanother embodiment, the amount of the alginate (including the peptide-grafted alginate) inthe aqueous bio-ink composition is 5.0%, based on the overall weight of the aqueous bio-inkcomposition, and a total of 1x103 to 1x108 cells per ml of the aqueous alginate solution. In yetanother embodiment, the amount of the alginate (including peptide-grafted alginate) in theaqueous bio-ink composition is 6.0%, based on the overall weight of the aqueous bio-inkcomposition, and a total of 1x103 to 1x108 cells per ml of the aqueous alginate solution.In one embodiment, the amount of the alginate (including the peptide-grafted alginate) in theaqueous bio-ink composition is 2.0%, based on the overall weight of the aqueous bio-inkcomposition, and a total of 1x106 to 5x107 cells per ml of the aqueous alginate solution. Inanother embodiment, the amount of the alginate (including the peptide-grafted alginate) inthe aqueous bio-ink composition is 3.0%, based on the overall weight of the aqueous bio-inkcomposition, and a total of 1x106 to 5x107 cells per ml of the aqueous alginate solution. In yetanother embodiment, the amount of the alginate (including the peptide-grafted alginate) inthe aqueous bio-ink composition is 4.0%, based on the overall weight of the aqueous bio-inkcomposition, and a total of 1x106 to 5x107 cells per ml of the aqueous alginate solution. In yetanother embodiment, the amount of the alginate (including the peptide-grafted alginate) inthe aqueous bio-ink composition is 5.0%, based on the overall weight of the aqueous bio-inkcomposition, and a total of 1x106 to 5x107 cells per ml of the aqueous alginate solution. In yetanother embodiment, the amount of the alginate (including the peptide-grafted alginate) inthe aqueous bio-ink composition is 6.0%, based on the overall weight of the aqueous bio-inkcomposition, and a total of 1x106 to 5x107 cells per ml of the aqueous alginate solution.The aqueous bio-ink composition of the invention can be reconstituted from a freeze-driedalginate-peptide product by adding distilled water or suitable buffer or a medium.In the second step of the method, the bio-ink composition is contacted with a crosslinkingsolution comprising a divalent and / or trivalent cation in the 3D printer to induce crosslinkingof the alginate. Preferably, the bio-ink composition is contacted with a solution comprisingCa2+, Sr2+, Ba2+, Cu2+, Zn2+, Mn2+, Fe2+, Cr3+, or Fe3+ cations. It is preferred herein to effectcross-linking by Ca2+ cations. Preferably, the cross-linking is achieved during the printing pro-cess by contacting the aqueous bio-ink composition with a solution containing the cation, e.g.,a 2%, 3% or 4% CaCl2 solution.The contacting of the bio-ink composition and the crosslinking solution can be carried out inthe print head of the 3D printer. In principle, any type of 3D printer is suitable provided thatviscous hydrogels can be printed with the device. A suitable printer preferably comprises aprint head with parametric flexibility that allows seamless adjustment of graft proportions with-out remodeling. The print head itself can be manufactured by 3D-printing using a clear resin.The print head is preferably designed to comprise a co-axial print head and two chambers, afirst inflow chamber for the crosslinking solution and a second inflow chamber for the bio-inkcomposition. The diameter of the first inflow chamber is between 1.5-10 mm, and preferablybetween 2.5 mm and 3.5 mm, and the diameter of the second inflow chamber is between 1.5mm and 2.5 mm, and preferably between 1.5 mm and 2.0 mm. In one embodiment, each ofthe two chambers are equipped with a Luer-Lock connector as an inlet and two nested tubesas outlets. Before the outlet, each chamber has a constriction serving as a resistance. Theresulting congestion homogenizes the flow of the bio-ink and ensures a uniform wall thick-ness. Additionally, immediately before the resistance, radially arranged lamellae are present,aligning the flow longitudinally. A suitable print head is exemplified in Figures 7a and 7b. Theaverage extrusion pressure is preferably set at 50-150 kPa, and preferably at 80-120 kPa,more preferably 100 kPa. The printing extrusion volume is preferably set at 5-50 ml / min, andpreferably at 10-30 kPa, more preferably 20 ml / min.In the last step of the method, the bio-ink is printed in the 3D printer into the bioartificial vas-cular graft. The printer that is used for carrying out the process preferably is a printer that iscapable of printing viscous fluids or hydrogels. Such a printer has a high extrusion power.The average extrusion pressure is preferably set at 150 kPa or more, and a co-axial printhead suitable for extruding viscous fluids or hydrogels. Additionally, such printer preferablyprovides adequate cubic capacity for printing a graft of 30-40 cm length or longer with preciseindustrial standard proceeding with a repeat accuracy of ^ 0.2 mm. The printer preferably issuitable of being used in in a clean room facility under GMP standards.The printing process can be carried out fully-automated without any manual intervention.Preferably, the printing process is performed in a sterile environment to provide GMP-com-pliant conditions. The bioartificial vascular graft printed in this way can have a length of up to100 cm length. In a preferred embodiment, the bioartificial vascular graft has a length between10 cm and 100 cm, and more preferably between 10 cm and 90 cm, between 10 cm and 80cm, between 10 cm and 70 cm, between 10 cm and 60 cm, between 10 cm and 50 cm,between 10 cm and 40 cm, between 10 cm and 30 cm, or between 10 cm and 20 cm. Abioartificial vascular graft having a length between 10 cm and 30 cm is particularly preferredherein. Moreover, branched vascular grafts can be fabricated as well by the method of theinvention, e.g. by using printer with a print head with a built-in branch or junction.The method of the invention for preparing a bioartificial vascular graft may further comprise astep (d) which comprises incubating the printed bioartificial vascular graft obtained from step(c) in a solution comprising a divalent and / or trivalent cation. This step may be helpful forachieving a complete alginate crosslinking. For this purpose, the bioartificial vascular graft isimmersed in a solution comprising the divalent and / or trivalent cation. The nature and con-centration of the cation solution used in this step can be as described above. Preferably, a2%, 3% or 4% CaCl2 solution can be used.The method of the invention for preparing a bioartificial vascular graft may also comprise astep (e) which comprises the further culturing of the graft obtained from step (c) or (d) in abioreactor to provide the complete differentiation of the cells on the grafts. Therefore, in apreferred embodiment, the bioartificial vascular graft obtained from step (c) or (d) is trans-ferred into a bioreactor for cell culturing. A standard culturing medium can be used, such asEndothelial Cell Growth Medium (ECGM, PromoCell, Heidelberg, Germany) which is supple-mented with 4 μL / mL of endothelial cell growth supplement, 0.1 ng / mL epidermal growth fac-tor, 1 ng / mL basic fibroblast growth factor, 90 μg / mL heparin, 1 μg / mL hydrocortisone (allfrom PromoCell) and 10% fetal bovine serum (Thermo Fisher, Dreieich, Germany). To complyGMP conditions, human AB serum can be used in a concentration of 2-10% instead of bovineserum. Another medium which can be used is the RPMI 1640 culture medium which is sup-plemented with 10% human serum. DMEM / Ham's 12 medium which is supplemented with10% human serum can also be used. Preferably, the cell culturing is performed for 12 hours,24 hours, 36 hours, 48 hours, 60 hours, 72 hours, or more. More preferably, the cell culturingis performed for at least 10 days, 14 days, 21 days, or longer. The cell culturing is preferablycontinued until a minimum cell number of about 1.0 x 106 cells / cm3, about 1.2 x 106 cells / cm3or about 1.5 x 106 cells / cm3 is achieved. Cell culturing is preferably performed under humidi-fied conditions at 37°C and 5% CO2.The invention also provides a bioartificial vascular graft obtainable by the above method. Thebioartificial vascular graft preferably is a tube or tube-like structure with a length as indicatedabove and with a minimum cell number of about 1.0 x 106 cells / cm3, about 1.2 x 106 cells / cm3or about 1.5 x 106 cells / cm3. Preferably, the graft has a mean total diameter of 2,000-4,000µm, and more preferably 2,500-3,500 µm, and an average wall thickness of 200-400 µm, andmore preferably 250-350 µm, which corresponds to average dimensions of small peripheralarteries and veins.The printed bioartificial vascular grafts can be used for a variety of medicinal and non-medic-inal purposes, including therapy, diagnostics, and drug screening. In addition, the printed bi-oartificial vascular grafts can be used as an in-vitro training model for endovascular or surgicaltraining.The invention hence provides a bioartificial vascular graft as described above for use in med-icine. In particular, the bioartificial vascular graft is for use in vascular or cardiovascular ther-apy. For example, the printed bioartificial vascular grafts can be therapeutically used, e.g., forvascular or cardiovascular bypass surgery, for vascular or cardiovascular vessel repair, or forthe preparation of a vascular or cardiovascular conduit after vessel trauma.The printed bioartificial vascular grafts can also be used as an in-vitro training model for endo-vascular or surgical training. The graft-based training model can be used for suturing, surgicalpreparation as well as catheter-based training. In addition, hematological and vessel-relateddiseases can be modelled and analyzed with the printed bioartificial vascular grafts of theinvention as an in-vitro model or in a lab-on-a-chip approach.The printed bioartificial grafts can also be used for the analysis of hematological and vessel-related diseases and for in vitro drug screening.BRIEF DESCRIPTION OF THE FIGURESFigure 1 shows the fabrication of the vascular grafts and design of the 3D bioprinting process.a) Endothelial Progenitor Cells (EPC) or Human Umbilical Vein Endothelial Cells (HUVEC)respectively are integrated into Collagen Peptide grafted Sodium Alginate (SA-COP) for thefabrication of the bio-ink. b) Immediate cross-linking of SA-COP by CaCl2 is conducted viathe co-axial printing process into support medium. c + d) 3D bioprinting is performed by thecustomized printing platform with an extrusion-based, co-axial print head. e) After bioprinting,the grafts are transferred to the bioreactor. f) Final design of the ready-to-implant vasculargrafts providing a vessel lumen between 3-3.5 mm and g) a total length of 30-40 cm. h) Flowand printing simulation of the print head were performed by the Ansys simulation software tocreate the print head prototypes i-iii. Print head iii represents the blueprint of the final printhead design and enables the printing of the finalized bioartificial graft composition (BGSA-COP20%). i) The reproducibility and holistic structure of vessel wall thickness, total diameter andlumen diameter were consistently refined during the simulation of the bioprinting process asshown by the performance results of the print head prototypes i-iii. Schematic illustration ofthe parametric print head used in the present examples. j) Schematic overview of the para-metric print head. k) Sideview of the parametric print head.Figure 2 shows the histological and immunohistological characterization of the 3D printedbioartificial grafts (BGSA-COP20%). a) Representative complete bypass cross section (HE stain-ing) with magnification (red rectangle). b) Elastica van Gieson (EvG) staining with magnifi-cation (red rectangle). c) Cell number per cross section in human Vein Grafts (VG) comparedto the cell number per cross section in the printed bioartificial grafts (BGSA-COP20%). d) 3D lifeimaging via fluorometric visualization with Calcein staining after 21 days of cultivation. e) MTSabsorbance (metabolic activity) of wet sample of Bioartificial Grafts from Sodium Alginate(BGSA), Bioartificial Grafts from Collagen Peptides grafted Sodium Alginate 2% (BGSA-COP2%)and 20% (BGSA-COP20%); *** = p < 0.001.Figure 3 Ultrastructural presentation of the Bioartificial Graft(s) from Collagen Peptidesgrafted Sodium Alginate (BGSA-COP). a + b) Surface of the bioartificial grafts and depiction ofthe fine network of interconnected fibers of BGSA-COP. c) Comparison of scaffold pore size (µm)between BGSA-COP 2% and BGSA-COP 20%. d) Comparison of porosity (%) between BGSA-COP 2%and BGSA-COP 20%.Figure 4 shows the results of the comparable biomechanical testing of human Vein Grafts(VG), Bioartificial Grafts from Sodium Alginate (BG-SA) and Bioartificial Grafts from CollagenPeptides grafted Sodium Alginate (BG-SA-COP). a) Simulation of small-vessel perfusion,burst strength and long-term resilience of BG-SA-COP in a flow model and sonographic con-trol of the grafts (red rectangle). b + c) Burst strength of VG, BG-SA and BG-SA-COP andYoung`s Modulus of VG and BG-SA-COP. d + e) Suture and tensile strength of VG and BG-SA-COP. f + g) Shrinkage Rate by Weight (SRW) and Swelling Ratio (SR) of BG-SA and BG-SA-COP; * = p < 0.05; ** = p < 0.01.Figure 5 shows the results of the assessment of coagulation and platelet activation. a) Chan-dler Loop model and exemplary depiction of thrombus (red rectangle). b.) Comprehensiveanalysis of thrombus size between human Vein Grafts (VG), Bioartificial Grafts from SodiumAlginate (BG-SA) and Bioartificial Grafts from Collagen Peptides grafted Sodium Alginate(BG-SA-COP) after 60 min of circulation in the Chandler Loop. c) Comprehensive analysis ofthrombus weight between VG, BG-SA and BG-SA-COP after 60 min of circulation in theChandler Loop. d) Total number of clots in the platelet-activation-assay after 15 min; * = p <0.05; ** = p < 0.01; surface of empty well was used as a control.Figure 6 shows the surgical implantation of the 3D printed Bioartificial Grafts from SodiumAlginate 20% (BGSA-COP20%) in a cadaver model (red rectangle a-c). a) Parachute anastomotictechnique was performed for cardiovascular graft implantation. b) Pull-down maneuver of thebioprinted vascular graft and c) final suturing of the graft creating a sufficient anastomosis inthe rabbit aorta.Figure 7 shows an overview on the novel 3D bioprinting technique of the invention.Figure 8 shows the results of the fluorescence imaging analysis. The results demonstrate thatthe bioink of the invention confers enhanced bioactivity and biocompatibility to the constructsproduced therewith.EXAMPLESThe examples described below were approved by the local ethics committee of the UniversityMedical Center Schleswig-Holstein, Kiel, Germany (protocol identification number: D518 / 13,D513 / 19 and D451 / 21 respectively). All procedures were performed in accordance with theHelsinki Declaration of 2013.Experimental groups comprised human saphenous Vein Grafts (VG), Bioartificial Grafts fromSodium Alginate (BG-SA), Bioartificial Grafts from Collagen Peptides grafted Sodium Alginate2% and 20% (BG-SA-COP-2 and BG-SA-COP-20). VG were obtained from voluntary donorsduring cardiac bypass surgery. The tissue quality of the VG and suitability for cardiovascularbypass grafting was assessed by duplex sonography and two cardiovascular surgeons notinvolved in the here described proof-of-concept study. Vascular grafts for the biomechanicalexperiments had a standard length of 60 mm and a standard diameter of 3.0-4.5 mm.Each experiment was repeated three times if not stated otherwise. All data are presented bythe Mean ± Standard Deviation (SD). Categorical variables are presented as frequency dis-tributions (n) and percentage (%). Data were analyzed by using Graph Pad Prism version9.2.0 (Graph Pad Software; San Diego, USA). Sample size calculation was performed by thefree available G*power Software and StatMate (Graph Pad Software; San Diego, USA). Nor-mality was tested by using the Shapiro-Wilk and Kolmogorov-Smirnov test. Data were ana-lyzed using one- and two-way ANOVA, Kruskal-Wallis-Test, or Student`s t test if appropriate.Tukey`s multiple comparison was performed as post-hoc-test. A p-value < 0.05 was consid-ered as significant.Example 1: Preparation of a bio-ink for 3D bioprintingA bio-ink was prepared by crosslinking a synthetic collagen-derived peptide (COP) that re-sembles the primary structure of collagen (N-(3-[2-Furyl]acryloyl)-L-G-P-A, available asFALGPA from SIGMA-Aldrich), and sodium alginate (SA) using the carbodiimide method.0.35 g SA was dissolved in 29 ml 0.2 M MES (2-(N-morpholino) ethanesulfonic acid) buffercontaining 0.3 M NaCl, pH 5.5. Then, 0.035 g N-hydroxy sulfosuccinimide (NHS) and 0.035g EDAC (1-Ethyl-3-(3ʹ-dimethylaminopropyl-carbodiimide, HCl) were added step by step intothe solution at room temperature with magnetic stirring for 20 min. The pH was slowly in-creased to 7.0 and the solution was set to a final volume of 35 ml (1% alginate solution). Forpreparing a bio-ink composition with 2% of COP coupled to SA (BGSA-COP2%) 3.5 mg of theFALGPA peptide was added into the solution. In a parallel approach, 35 mg of the FALGPApeptide was added into the solution to prepare a bio-ink composition with 20% of COP cou-pled to SA (BGSA-COP20%). The coupling reaction was allowed to proceed for 7-8 h under stir-ring.The two solutions so produced were purified by dialysis through a 6-8 KD molecular weightcut-off dialysis tube for three days. The dialyzed products were finally freeze-dried with Ly-oVac GT2 freeze dryer to obtain purified alginate derivative powders. The dried samples werestored at -20°C. Freeze-dried alginate products without and with coupled collagen peptidewere reconstituted to 4% alginate with sterile distilled water. The coupling of peptide to algi-nate was confirmed by measuring the differences in the absorbances compared to an alginatecontrol. The increase in absorbance by the peptide was calculated as followed:Increase of absorbance (%) = [Abs (SA-COP) – Abs (SA)] / Abs (SA) x 100The presence of peptide increases the absorption of UV light at 200 nm (for peptide bonds),at 280 nm (for aromatic rings) and at 345 nm (for FALGPA) as described previously (Jacksonet al., 1995; Morch et al., 2007).Results: Different bio-ink compositions were produced. Specifically, bio-ink compositions withlow (2%) and high concentration (20%) of COP coupled to SA were used for the bioprintingof bioartificial grafts (BGSA-COP2%, BGSA-COP20%) and compared to sole SA manufactured grafts(BG-SA) and human Vein Grafts (VG) to investigate the biomimetic characteristics.Example 2: Preparation of a cell-containing bio-inkThe final bio-ink for use in 3D bioprinting was prepared by adding Human Umbilical VeinEndothelial Cells (HUVEC) or Endothelial Progenitor Cells (EPC) to the matrix obtained fromExample 1. HUVEC were isolated from umbilical cords and cultured in endothelial cell growthmedium ECGM (PromoCell, Heidelberg, Germany) supplemented with 4 μL / mL of endothelialcell growth supplement, 0.1 ng / mL epidermal growth factor, 1 ng / mL basic fibroblast growthfactor, 90 μg / mL heparin, 1 μg / mL hydrocortisone (all from PromoCell) and 10% fetal bovineserum (Thermo Fisher, Waltham, USA) (Baudin et al.2007). A suitable protocol for the isola-tion of HUVEC is described in (Crampton et al. 2007). For complying with GMP conditions,the bovine serum can be replaced by 2-10% human AB serum.EPC were obtained from whole blood samples (40 ml) of healthy volunteers after informedconsent and in accordance with the local ethics committee (see the protocol in: Mead et al.,2008). Briefly, whole blood was mixed Mix 1:1 with Ca2+-Mg2+-free PBS and blood mononu-clear cells were isolated using Ficoll density gradient centrifugation according to the manu-facturer’s protocol (GE Healthcare, Chicago, USA), followed by washing with phosphate-buff-ered saline (PBS). Further isolation of EPC was performed by CD34+ cell-sorting via mag-netic beads according to the manufacturer’s protocol (Miltenyi, Bergisch Gladbach, Ger-many). Cells were then plated in collagen-coated flasks and cultured with endothelial basalmedium 2 (EBM-2; Lonza, Basel, Switzerland). Characterization of EPC was performed bymorphological analysis and flow cytometry. Briefly, flow cytometry was performed using theBD FACS Calibur™ cytometer (BD Biosciences, Franklin Lakes, USA). Specific antibodiesand their corresponding isotypes (dilution for all antibodies: 1:20) were directly conjugatedwith fluorescein isothiocyanate (FITC), alexa fluor, or allophycocyanin (APC)-Cy7. The gat-ing strategy consisted of (i) identification of EPC based on their size and granularity (FSC vsSSC profiles), (ii) exclusion of non-viable cells and (iii) identification of EPC positive for CD31,CD34, CD146, CD309 and negative of CD45 and CD14.HUVEC or EPC were detached, counted and maintained in cell culture medium before prep-aration of the final bio-ink formulation. The desired calculated cell quantity was carefully mixedwith the 4% SA-COP solutions to obtain final cell concentration in the bio-ink of 2.5 x 106cells / ml and 3% of SA-COP in the final bio-ink.Results: The bio-ink comprising SA-COP and the EPCs resembled the physical compositionof a viscous gel at room temperature.Example 3: 3D bioprinting of vessel-like structureSince most of the available commercial 3D bioprinting systems do not provide adequate cubiccapacity for printing a vascular graft of 30-40 cm length (Figure 1f + g), a customized 3Dbioprinting platform was used in order to obtain a homogenic outflow in axial direction for theextrusion of the high-viscosity bio-ink (Figure 1c + d). The Ansys CFX 2020 R2 simulationsoftware (Ansys, Canonsburg, USA) was applied for prototyping of the print head design(Figure 1h + i; print head prototype i-iii) and the simulation of the printing process itself.Viscosity was set at 8.3 Pa*s and density was set at 1.79 g·cm−3 for alginate during the sim-ulation process. The final print head design (Figure 1h + i; print head iii) included a maininflow chamber for the CaCl2 support medium for immediate cross-linking and a lateral inflowchamber running the cell-loaded bio-ink (Figure 1c). It is commonly accepted that immediateand rapid cross-linking of microfibers during the printing process is mandatory for the laterresilience of the fabricated grafts. The central inflow chamber conducted the 4% CaCl2 sup-port medium for rapid cross-linking and the lateral inflow chamber provided the cell-loadedbio-ink with an average flow of 20 ml / min (Figure 1c). The printing process was performed atroom temperature, pressure-controlled and extrusion-based due to the high viscosity of thebio-ink. Average extrusion pressure was set at 100 kPa with a printing speed of 20 ml / min.The co-axial print head was guided by a 6-axis robot arm (Stäubli TX2-40 6-axis robot;Stäubli, Pfäffikon, Switzerland) enabling precise proceeding with a repeat accuracy of + / - 0.2mm, six degrees of freedom and a sterile printing process without potential contamination.The BGs were printed into support medium of 4% CaCl2 for additional cross-linking (Figure1d).Results: Advanced simulation of the in- and outflow characteristics of the co-axial printingprocess significantly improved and homogenized graft morphology (Figure 1h + i + j + k).Reproducibility of the bioprinting process improved significantly after computational flow sim-ulation and was accordingly high in the final configuration (e.g., standard error of wall thick-ness of the vascular grafts < 10%). The final BGs had a mean total diameter of 3092 ± 307.7µm, a lumen of 2472 ± 275.6 µm and average wall thickness of 307.2 ± 67.97 µm which is inline with average dimensions of small peripheral arteries and veins. The final 3D bioprintingplatform enabled the fabrication of a BG with a length of 30-40 cm which is comparable witha human saphenous vein from the lower or upper leg commonly used as a VG for cardiovas-cular surgery (Majesky et al., 2018; Kentenciller et al., 2018) (Figure 1f + g). These dataclearly suggest that the grafts prepared according to the invention are suitable for use inhuman medicine (Figure 1 + 2).The crosslinking of the bio-ink by CaCl2 leads to a shrinkage of the bypass graft during themanufacturing process. Additionally, the bio-ink exhibits non-Newtonian fluid behavior. Bothfactors necessitate the incorporation of an offset into the parameters for the diameter andwall thickness of the grafts to achieve the target parameters. The size proportions of theproduced graft were carefully measured under a microscope and compared to the target pa-rameters. Over three iterations, an optimal offset for the diameter and wall thickness param-eters was determined (Figure 1j +k). The following Table 1 presents the results for the graftand print head parameters used in this study:Target parameter Print head parameterDiameter 4 mm 5.2 mmWall thickness 0.8 mm 1.5 mmLumen 2.4 mm 2.2 mmTable 1: Results for the graft and print head parameters used in this study.Example 4: Cultivation of the vascular grafts in the bioreactorAfter printing, the vascular grafts remained 6 h in 4% CaCl2 and culture medium (1:1) andwere then cultivated for 72 h in a rotating cell seeder (Aptus Bioreactors, Clemson, USA), abioreactor (Figure 1e) with a slightly modified protocol (Movileanu et al., 2021). Briefly, thebioreactor settings were chosen by experimental pre-testing to enable a continuous flow dur-ing reactor movement, regular rest periods for advanced cell encapsulation and preventionof exceeded wall-attachment. After a period of static immersion in culture media for 1 h thebioreactor was rotated at 40º and 60º respectively and at 3 rotations per minute (RPM). Therewas a programmed rest period for 10 min after each cycle. For this dynamic cell seeding, anair pump was attached to the rotator jars via a sterile inline filter, continuously enriching theculture media with air from the CO2 incubator (Movileanu et al., 2021). After the cell seeding,the grafts were transferred to conventional 3D cell culture. The vascular grafts were culturedfor a total of 21 days in order to reach a minimum estimated cell number of approximately 1.6x 106 cells / cm3. Cultivation was performed under humidified conditions at 37◦C and 5% CO2.Example 5: Analysis of cell viability, density, and proliferationThe cultured grafts obtained from Example 4 were analyzed by fluorometric visualization todetermine cell viability, density and proliferation after 21 days of cultivation. Living cells werequalitatively assessed by fluorometric visualization. Small pieces (about 0.5 cm) of the bio-ink were removed and added in another well with fresh medium containing 0.1 µM CalceinAM (Thermo Fisher, Waltham, USA) and 2 µg / ml Hoechst 33342 (Invitrogen, Carlsbad, USA)for the assessment of cell viability and cell nuclei, respectively. Staining of cells was left for30 minutes inside of the incubator in the dark. A small piece of bio-ink was mounted betweena glass slide and a coverslip. Live cells were visualized using a Leica microscope (LeicaDM2000, Leica, Wetzlar, Germany) with fluorescent filters. Cell numbers and densities (nu-clei / cm2) were evaluated in fluorescent images of Hoechst 33342-stained cells using the au-tomatic image-based tool for counting nuclei (ITCN) from the Image J software 1.41 (NIH).The metabolic activity and cell proliferation within the vascular graft were evaluated after 21days of cultivation by using a colorimetric kit (CellTiter96® AQueous One Solution ReagentG3580 Promega Madison, WI, USA). Small specimens (about 1 cm in length) of the vasculargraft were removed and added into a new culture plate with 1 ml of fresh culture medium.200µl of MTS reagent was applied to each well and culture was continued at 37°C. Metabolicactive cells release a colored product into the culture medium that was evaluated by absorb-ance at 490 nm using an ELISA reader (Tecan, Crailsheim, Austria) in combination with theMagellan software v1.1. Every hour between 1-10 hours of incubation after the addition ofMTS reagent, 100µl of culture media was taken at each time point and analyzed. At the endof the experiment, the BG specimen used to evaluate metabolic activity by MTS was re-moved, let dry completely at 95°C for 48h and weighed. For the graphic, the absorbance pergram of wet sample was depicted at each time point. The metabolic activity per sample ateach time point was calculated as follow:Metabolic activity: (MTS Abs490nm / grams of wet sample)Samples of the vascular grafts were furthermore visualized by Live-3D-Cell-Imaging using aLeica THUNDER Imager 3D assay (Leica Microsystems GmbH, Wetzlar, Germany) basedon Leica DMi8 microscope with 5x objective lens (PLAN 5x / 0.12 dry), 20x objective lens (HCPL APO 20x / 0.80 dry) and Leica K5 camera (4.2 MP, 40 FPS). Cool LED pE4000 was usedas a fluorescence light source and the filter cubes with the desired excitation and emissionswere selected (DAPI or LED-405 EX: 405 / 60, EM: 470 / 40; or FITC EX:480 / 40, EM:527 / 30 orTXR EX:560 / 40, EM:630 / 75). Normal or top to bottom Z-stack images of the samples wereacquired using the same set of filters and settings (intensity, exposure time, threshold and Z-step size). Images were processed with Leica LAS X software and the Small Volume Com-putational Clearing (SVCC) was applied. Immunofluorescence staining was analyzed usingthe Image J software 1.41 (National Institutes of Health NIH) and cell number in 3D cell culturewas analyzed layer-by-layer. Total cell number for the whole vascular grafts was finally cal-culated based on a thickness of 4 µm for every specimen and a total of 15000 layers withinthe cultured vascular grafts respectively. Specimen of human native veins served as controlgroup.Samples of the vascular grafts were also fixed in 4% formalin and embedded in paraffin. Six4 μm thick sections from the proximal, middle and distal end were prepared and routinelystained with Haematoxylin-Eosin (HE), Elastica van Gieson (EvG) and Sirius Red. Immuno-histochemical CD31 staining was performed with an automated Bondmax staining system(Leica Biosystems, Wetzlar, Germany) using the Polymer Refine Detection Kit (Leica Biosys-tems) and anti-CD31 antibody (clone JC70, Cell Marque, Rocklin, California, United States)at a dilution of 1:100.Results: After initial bioprinting and cultivation in the bioreactor, cell density, viability and cellnumbers were qualitatively assessed by fluorometric visualization (Figure 2d). Excellent via-bility and significant higher cell density could be demonstrated in SA-COP in contrast to SA.After 21 days in culture, endothelial cells within the bioartificial vessel wall showed continuousmetabolic and a significant higher proliferative activity in BGSA-COP20% (Figure 2e). It is well-known that alginate does not provide any cell-binding motifs in contrast to collagen. Thus, theincrease of the proportion of collagen in SA-COP hydrogels enables significant more celladhesion and thereby maintains cell viability and proliferation. It is also likely that COP couldstimulate the synthesis of extracellular matrix components and consequently speed up tissueformation and promote cell adhesion and proliferation (Fan et al., 2013; Ullah et al., 2020; Huet al., 2021).Further immunobiological staining of the BG-SA-COP demonstrated strong expression of theendothelial surface marker CD31. Collagen-specific analyses employing Elastica von Gieson(EvG) and Sirius Red staining confirmed the presence of a dense matrix (Figure 2b). After acultivation period of 21 days, Calcein AM staining revealed high cell viability and a tendencyfor cellular organization in the evolved graft as demonstrated by Live-3D-Cell-Imaging (Figure2d). Total cell number in specimens of BG-SA-COP was slightly lower than in control samplesof VG but without statistical significance (Figure 2c). Calculation of the total number of viablecells per vascular graft yielded approximately 2.1 x 107 cells and was comparable to speci-mens of VG with an estimated total cell number of 2.6 x 107 (graft dimensions: 60 mm x 3.0 -4.5 mm).Taken together, advanced morphological examination of the BGSA-COP20% revealed a tendencyof cellular organization and homogenous artificial tissue growth within a collagen-based ma-trix which suggest biomimetic characteristics of the fabricated vascular grafts.Example 6: Analysis of porosity and pore sizeThe cultured grafts obtained from Example 4 were further analyzed by Scanning ElectronMicroscopy (SEM) to determine pore size and tissue network of the vascular grafts. Vesselspecimens were fixed in 2.5% glutaraldehyde and postfixed in 2% osmium tetroxide, irrigatedwith distilled water and then dehydrated in series of 50–100% ethanol. The specimens werethen placed in 100% ethanol and dried by critical point dryer (Balzers, CPD030) and mountedonto SEM slabs and sputter-coated for 50 seconds, 38mA with gold by sputter coater(Balzers, SCD050). Examination of the vessel specimens was performed with a JSM-IT200(JEOL, Tokyo). The Image J software was used to analyze SEM images to assess mean poresizes and to establish pore size distributions in the samples. Porosity was measured via aliquid substitution approach using the following formula and as described before (Xu et al.,2021): p (%) = (V1-V3) / (V2-V3), where p corresponds to porosity, V1 denotes the startingvolume of ethanol, V2 denotes the volume of ethanol following sample immersion for 10 min,and V3 denotes the residual volume following wet sample removal.Results: Scanning Electron Microscopy (SEM) imaging revealed a porous and well-intercon-nected microstructure as seen in Figure 3a + b. Pore size for embedded cells or cell debrisranged from 10.40-81.50 µm in BGSA-COP 2% and 9.08-78.30 µm in BGSA-COP 20% and from 0.11-0.42 µm in BGSA-COP 2% and 0.17-0.43 µm in BGSA-COP 20% for the surface of the bioartificialvessel wall (Figure 3c). Both BGSA-COP were highly porous with porosity values as demon-strated in Figure 3d. As described before the degree of porosity and refining is crucial forintegration of biomaterials determining cell seeding, proliferation and cell metabolism [25, 26,27]. As described before the degree of porosity and refining is crucial for integration of bio-materials determining future cell seeding, proliferation and cell metabolism (Tamaddon et al.,2017; Xu et al., 2021; Su et al., 2021). High-porosity scaffolds (e.g. > 70% porosity) indicategood nutrition supply and neo-vascularization enabling stable long-term results after in-vivoimplantation. Generally, a pore size range of 10-500 µm is considered as beneficial for scaf-fold integration which is in common with the here described experimental evaluation of bothBG-SA-COP (Xu et al., 2021; Su et al., 2021).Example 7: Analysis of biomechanical characteristicsThe cultured grafts obtained from Example 4 were further analyzed for their biomechanicalcharacteristics. Burst strength of VG, BG-SA, BGSA-COP2% and BGSA-COP20% were comparedusing a previously described method (Clements et al., 2007). Briefly, the grafts were fixed ina customized pressure chamber filled with PBS simulating tissue environment and serving asleakage control. The distal ends of the grafts were sealed, and proximal ends were perfusedby a peristaltic pump infusing PBS at a constant rate of 50ml / min until graft burst. The highestpressure measured before failure was defined as the burst strength value.For determining the elasticity of the grafts, Young`s Modulus was measured. The Young’smodulus (E) represents a material property and its capability for stretch and deformation. Eis defined as the ratio of tensile stress (σ) to tensile strain (ε). Where stress is the amount offorce applied per unit area (σ=F / A) and strain is extension per unit length. E was calculatedusing the following equations: ^^ ∕ ^^ == ^^^ ∕ ^Tensile and suture strength characteristics of VG and BG-SA-COP were evaluated in a stand-ard tensile test station (Z 0.5 Zwick / Roell, Ulm, Germany). Each vascular graft was mountedon an irregular polymer surface to prevent slippage during the test series (Fixation zone ofeach graft: 5 mm; Figure 4d + e). After applying the mechanical force (1 mm / min), vasculargrafts ruptured near the middle or near both ends. Force measurement was recorded by au-diting software testXpert II (Zwick / Roell, Ulm, Germany). For tensile strength testing, a stand-ard synthetic, non-absorbable, monofilament surgical suture (Prolene monofil FS26-0, Ethi-con, Cincinnati, USA) was placed 3 mm towards the edge of the vascular graft. Both experi-mental procedures have been described previously (Clements et al., 2007; Konig et al., 2009;Quint et al., 2012; Hoganson et al., 2018).Results: The systematic comparison of burst strength between VG (1.82 x 105 ^ 0.29 Pa),BG-SA (1.47 x 105 ^ 0.22 Pa), BGSA-COP2% (1.72 x 105 ^ 0.19 Pa) and BGSA-COP20% (1.97 x 105^ 0.24 Pa) revealed significant higher burst strength for BGSA-COP20% compared to sole SAfabrication (p = 0.00) (Figure 4a + b). Hence, the biomechanical capabilities of both BG-SA-COP for maximal pressure exposure were similar to human VG in this model which is in linewith other studies that have described burst strength for vascular grafts ranging between 0.5- 2.6 x 105 Pa (Clements et al., 2007; Konig et al., 2009; Quint et al., 2012; Hoganson et al.,2018).For elasticity analysis of BG-SA-COP, Young`s Modulus was determined which was compa-rable between human VG and both BG-SA-COP (Figure 4c). Calculation of Young`s Modulusrevealed 771.7 ^ 143.7 kPa for VG, 710 ^ 130.4 kPa for BGSA-COP2% and 761.7 ^ 140.8 kPafor BGSA-COP20%, respectively (Figure 4c). Consequently Young`s Modulus of BG-SA-COP wascomparable or appreciably higher than in previous studies examining BG-SA and the elastic-ity of both BG-SA-COP appeared comparably to human-scale vessels (Clements et al., 2007;Konig et al., 2009; Quint et al., 2012; Zhang et al., 2015; Hoganson et al., 2018) (Figure 4c).Moreover, suture strength of BG-SA-COP was lower but still comparable to the performanceof human VG in the test station (Figure 4d). Suture strength was higher in the VG group (0.65^ 0.10 N) compared to the BGSA-COP2% (0.33 ^ 0.16 N) and BGSA-COP20% group (0.41 ^ 0.25 N)but only VG and BGSA-COP2% differed significantly (p = 0.02) (Figure 4d). The reduced suturestrength of both BG-SA-COP might be the result of reduced interconnection of fibers in theend sections of the grafts as a consequence of the current fabrication process. Further im-provement of this technical issue could be potentially addressed by compression and spin-ning technologies for fibrin and collagen constructs as reported by various authors. Extensionof cross-linking or detaching the end section of the graft could also address this aspect (Helmset al., 2021).Tensile strength was also similar between VG (8.96 ^ 1.02 N) and BGSA-COP2% (8.24 ^ 1.74 N)respectively BGSA-COP20% (9.46 ^ 2.60 N) (Figure 4e). It should also be mentioned that BG-SAbroke after few seconds in the test station and therefore no sufficient measurement was pos-sible. Both suture and tensile strength experiments with BG-SA were therefore excluded fromthe remaining experiments.Overall, the biomechanical key parameters were comparable between BGSA-COP20% and VG.The fact that the biomechanical test results for VG are comparable to formerly publishedstudies underline the robustness of the here described experimental set-up (Clements et al.,2007; Konig et al., 2009; Quint et al., 2012; Hoganson et al., 2018).Example 8: Analysis of dehydration and swellingThe cultured grafts obtained from Example 4 were further analyzed for their dehydration andswelling characteristics. For this purpose, BG-SA and BG-SA-COP were soaked in 4% CaCl2and culture medium (1:1) for 6 h followed by a dehydration step at room temperature for fourdays (Zhang et al., 2015). The dehydrated vascular grafts were then soaked in PBS (SigmaAldrich, U.S.A.) for the evaluation of swelling, Shrinkage Rate by Weight (SRW) and SwellingRatio (SR). SRW and SR were calculated using the following equations and as describedbefore (Zhang et al., 2015): ^^ =^^^^^ ^^ ^100% (2)W0 represents the original weight after fabrication, Wi is the swollen weight at the predeter-mined time point and Wd is the dehydrated weight.Results: The test revealed that lumen and total dimension of the grafts were mostly preservedafter the dehydration process (Figure 4f). However, BGSA-COP2% lost more weight as indicatedby the Shrinkage Rate by Weight (SRW) (83.78% ^ 12.31%) over time than both BG-SA(81.77 ^ 8.10%) and BGSA-COP20% (66.48% ^ 6.27%) (BGSA-COP2% vs BGSA-COP20%, p = 0.03)(Figure 4f). The SRW for the BG-SA group was also comparable to 3-4% alginate tubes de-scribed by Zhang and co-workers (Zhang et al., 2015). These findings suggest that SRWdecreases with increased COP concentrations. A relatively low SRW also suggests a stableorganization on macromolecule level and functional cross-linking which is highly favorable forthe here demanded scope of application (Zhang et al., 2015).The swelling experiments followed the initial dehydration: in accordance, the Swelling Ratio(SR) decreased with increasing proportion of COP in BGSA-COP (BGSA: 2399.19% ± 691.63%;BGSA-COP2%: 1844.42% ± 261.06%; BGSA-COP20%: 1326.33% ± 443.03%) respectively (Figure4g). However, only the comparison between BGSA and BGSA-COP20% differed significantly (p =0.01) highlighting the incorporation of collagen in the bio-ink as a significant structural element(Figure 4g).Example 9: Analysis of coagulation activation and thrombogenicityTo analyze coagulation characteristics of the printed BG-SA-COP, two established modelswere used: first coagulation activation and solid thrombus formation by whole blood was in-vestigated in the dynamic Chandler Loop model and second platelet activation was analyzedby applying Platelet Rich Plasma (PRP) in a platelet-activation-assay (Olsen et al.2018).To analyze coagulation activation by the bioprinted grafts, the established Chandler Loopsystem was used in a slightly modified setting (Olsen et al.2018). Briefly, in this dynamic in-vitro model, the vascular grafts were integrated into silicon tubes and filled with whole bloodfrom four donors who underwent full coagulation diagnostics prior to the experiments. Thevascular grafts then rotated in a constantly warmed water bath at 37^C and maximal clottingformation was assessed by weighing and measuring the thrombus at 30 and 60 min.Besides full coagulation activation by BG-SA-COP, the distinct activation of platelets by SAand SA-COP was assessed by a platelet-activation-assay applying Platelet Rich Plasma(PRP). 200 μL of sole SA, SA-COP2% and SA-COP20% were placed on the bottom of a 12well plate. A solution of 50 % citrated blood and CaCl2 (2mM) was prepared, vortexed for 20s and centrifugated at 1800 x g for 10 min. The obtained blood plasma was again centrifu-gated four times at 1800g to receive PRP. Afterwards, 200 μL of PRP were placed into eachwell. The control group was composed only by PRP and therefore without surface activationof thrombocytes by SA or SA-COP. The wells were washed with PBS solution at 5, 10 and15 min to stop clotting and remove soluble clots. Clotting time was defined after first formationof solid clots and number of clots was visually assessed via microscopic examination.Results: It was found that after 30 min in the Chandler Loop, only the BG-SA showed distinctthrombus formation indicating a higher thrombogenicity than the other experimental groups.After 60 min thrombi occurred in all four experimental groups. No major differences in throm-bus weight and size between VG, BGSA-COP2% and BGSA-COP20% were observed in these exper-iments (VG: weight-thrombus = 8.62 ^ 0.88 g; size-thrombus = 29.83 ^ 3.06 mm; BGSA-COP2%:weight-thrombus = 8.23 ^ 0.85 g; size-thrombus = 28.33 ^ 1.63 mm; BGSA-COP20%: weight-thrombus = 7.73 ^ 0.58 g; size-thrombus = 29.50 ^ 3.93 mm) and only comparison of throm-bus weight between the BG-SA and BGSA-COP20% differed significantly (p = 0.03) (Figure 5a-c). Overall, BGSA-COP2% and BGSA-COP20% showed a similar performance in in-vitro thrombo-genicity compared to human VG. It has been described that exposure of extracellular matrixproteins especially fibrillar collagens I and III are required for thrombus formation and plateletadhesion (Smethurst et al. 2007). However, there was no significant difference in in-vitrothrombogenicity between both BG-SA-COP groups despite the higher percentage of poten-tially thrombogenic COP in BGSA-COP20%.The platelet-activation-assay also supported the results from the Chandler Loop model: therewas not a significant difference in both SA and the SA-COP groups indicating no enhancedplatelet activation by the rising percentage of COP (Figure 5 d). These findings could be dueto the highly aligned ultrastructure of the grafts with metabolically active endothelial cells pre-venting thrombus formation and platelet activation. Although it is commonly accepted thatthrombogenicity is positively influenced by the presence of collagen, the thrombus formationwas more pronounced in BG-SA than in BG-SA-COP in the Chandler Loop model.These findings indicate that the properties of collagens, particularly with regard to thrombo-genicity, are modified by combination with SA and that the resulting structure has differentphysical and physiological properties than the original substances alone. The results obtainedalso confirm the suitability of the grafts produced for putative surgical implantation.Example 10: Endurance and permeability testing in an in-vitro circulation modelPermeability and initial stability characteristics of the printed vascular grafts were initially as-sessed by implantation into an established slightly modified in-vitro circulation model consist-ing of a circulatory pump enabling a consistent circulation of 500 ml NaCl 0.9% fluid temperedat 37°C (Rusch et al., 2019). The vascular grafts were implemented in the circulation modelvia ligature with vessel loops (Primed, Halberstadt, Germany). A consistent flow rate of 80ml / min and 100 mmHg simulated the physiological circulation in the coronary artery or smalldiameter vessels of the lower limb for 5 d (Westerhof et al., 2006). Fluid was collected in aretention basin and flow was consistently assessed via laser doppler flowmetry. After theexperiments, the vascular grafts were examined via microscope.Results: In-vitro testing revealed a low average leakage rate of approximately 0.83 % in theBGSA-COP2% and 0.57 % in the BGSA-COP20% group after 5 d in the circulation model (Figure 4a).These results were comparable to an average leakage rate of 0.46 % in the VG group. Thehigher leakage rate in the BGSA-COP2% might result from the lower level of interconnected fibresand greater pore size as demonstrated in the SEM imaging (Figure 3a + b). Microscopicinspection did not reveal any structural damage of the grafts after 5 d (data not shown).Example 11: Surgical implantation in a perfused cadaver modelFor initial assessment of surgical suitability of the bioprinted grafts, implantation of the finalBGSA-COP20% in Wistar rats (n = 3) and New Zealand rabbits (n = 3) were performed. Implan-tation of VG served as control group. Two cardiovascular surgeons performed the end-to-endimplantation of the grafts as partial replacement of the aorta. Anastomosis were performed inongoing suturing and parachute technique with Prolene monofil FS26-0 (Ethicon, Cincinnati,USA) (Figure 6a-c). After surgery, permeability of the anastomosis and grafts was tested viaon-pump perfusion of the animal aorta with approximately 80 ml / min simulating coronary orcrural arterial supply. Evaluation of the completed anastomosis was assessed by applyingthe slight modified Northwestern Objective Microanastomosis Assessment Tool (NOMAT) fo-cusing only on the sufficiency and quality of anastomosis (Items XII-XIV, range 0-15), sub-jective (range 0-100) and in-vitro performance grading (pass / fail) made by two independentsurgical trained observers via video of the procedures (Table 2) (Aoun et al., 2015).Results: It was found that surgical implantation in two different perfused cadaver models (NewZealand rabbit n = 3; Wistar rat n = 3) was performed without technical issues even in de-manding parachute anastomosis technique (Figure 6a-c). Only one graft failure was observeddue to suture tearing. Grading of the performed surgery via the Northwestern ObjectiveMicroanastomosis Assessment Tool (NOMAT) for surgical handling and sufficiency of thevascular anastomosis was comparable to surgery performed with human VG (Table 2). Ac-cordingly, inspection of the anastomosis did not show any sign of high-grade stenosis orleakages after implantation.Whereas most described tissue-engineered vascular grafts have been acelluar vascularprothesis or preliminary approaches without surgical feasibility, this is the first time that abioprinted bioartifical vascular graft has been successfully implanted in a surgical model andfulfils all relevant real-world benchmark parameters (Dahl et al., 2011; Zhang et al., 2015;Hong et al., 2019; Cui et al., 2019).SA-COP 20% grafts human Vein GraftsMean NOMAT (range) 12.3 (9-15) 13.5 (11-15)Mean subjective grade (range) 70.6 (55-84) 69 (55-80)In-vitro performance 5 pass, 1 fail 6 pass, 0 failedMean anastomosis time in min (range) 16.4 (15.4-18.4) 13.75 (13.4-18.2)Table 2: Results of the implementation of the final vascular grafts in the cadaver model(n = 6). Performance data of the Northwestern Objective MicroanastomosisAssessment Tool (NOMAT) grading for the evaluation of anastomoses andgraft handling for BGSA-COP20% in contrast to human Vein Grafts (VG).Example 12: Bioink Composition including peptides derived from collagen, elastin, and lam-inin coupled to alginateAn improved bio-ink formulation was prepared by coupling alginate with collagen peptidesand integrating the extracellular matrix (ECM) components - laminin and elastin - as hybridpeptides. The carbodiimide reaction was used to mediate the crosslinking between sodiumalginate and the following ECM-derived peptides: the laminin-elastin deca-hybrid peptideNH2-K-S-I-R-V-G-V-G-P-G-COOH (SEQ ID NO:8), obtained from ABI scientific, and the col-lagen pentapeptide N-(3-[2-Furyl] acryloyl)-L-G-P-A obtained from SIGMA-Aldrich) .0.35 g sodium alginate was dissolved in MES (0.2 M MES (2-(N-morpholino) ethanesulfonicacid) containing and 0.3 M NaCl, pH 5.5). Then, 0.035 g N-hydroxy sulfosuccinimide (NHS)and 0.035 g N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) wereadded step by step into the solution at room temperature with magnetic stirring for 20 min.The pH was slowly increased to 7 and the peptides (0.035g) were added into the solutionfacilitating the coupling for 8 h. The produced 1% alginate derivative solution was purified bydialysis through a 6-8 KD molecular weight cut-off dialysis tube for three days. The resultingNH2-K-S-I-R-V-G-V-G-P-G-COOH and N-(3-[2-Furyl] acryloyl)-L-G-P-A containing solutionswere then combined in a 1:5 ratio to create the improved bioink composition. The improvedbioink was finally freeze-dried in a LyoVac GT2 to obtain a purified alginate derivative powder.The dried samples were stored at -20°C.Prior to application, the improved bio-ink was re-suspended in distilled water to achieve aconcentration of 5% alginate. Cryopreserved endothelial progenitor cells (EPCs) or endothe-lial cells, respectively, were thawed and cultured for 24 hours. On the day of use, cells weredetached and incorporated into the improved bio-ink to achieve a final concentration of 4%alginate derivative containing approximately 1 million EPCs per milliliter. The improved bio-ink formulation was then employed for 3D bioprinting of bioartificial vessels with a diameterof 3.5 mm and a wall thickness of 0.7 mm.In addition, the bio-ink was tested for its ability to support and optimize the spatial arrange-ment, structural integrity, and viability of EPCs embedded within the 3D printed constructs.Fluorescence imaging was employed to assess cell viability following 5 days of incubation.Specifically, Calcein AM staining (green) was used for selectively labeling vital cells, andHoechst 33342 staining (purple) was used to label the nuclei.Results: Fluorescence imaging confirmed high cell survival rates in the constructs. The ob-served staining pattern (see Figure 8) proves that a substantial proportion of the cells re-mained viable throughout the culture period. These findings suggest that the optimized bio-ink composition not only supports cell viability, but also facilitates the fabrication of mechani-cally stable and biocompatible bioartificial vascular structures. Such structures demonstrategreat potential for use in regenerative applications, where both functional and structural in-tegrity are essential.LITERATURE1. Kullo, I.J. and Rooke, T.W., 2016. Peripheral artery disease. New England Journal ofMedicine, 374(9), pp.861-8712. Head, S.J., Milojevic, M., Taggart, D.P. and Puskas, J.D., 2017. Current practice ofstate-of-the-art surgical coronary revascularization. Circulation, 136(14), pp.1331-1345.3. Caps, M.T., 1998, December. The epidemiology of vascular trauma. In Seminars invascular surgery (Vol.11, No.4, pp.227-231).4. Haney, L.J., Bae, E., Pugh, M.J.V., Copeland, L.A., Wang, C.P., MacCarthy, D.J.,Amuan, M.E. and Shireman, P.K., 2020. Patency of arterial repairs from wartime ex-tremity vascular injuries. Trauma surgery & acute care open, 5(1), p.e0006165. Caliskan, E., De Souza, D. R., Boening, A., Liakopoulos, O. J., Choi, Y. H., Pepper,J., ... & Emmert, M. Y. (2020). Saphenous vein grafts in contemporary coronary ar-tery bypass graft surgery. Nature Reviews Cardiology, 17(3), 155-169.6. Gallo, M., Trivedi, J. R., Monreal, G., Ganzel, B. L., & Slaughter, M. S. (2020). Riskfactors and outcomes in redo coronary artery bypass grafting. Heart, Lung and Cir-culation, 29(3), 384-389.7. Xenogiannis, I., Zenati, M., Bhatt, D. L., Rao, S. V., Rodes-Cabau, J., Goldman, S.,(...) & Brilakis, E. S. (2021). Saphenous vein graft failure: from pathophysiology toprevention and treatment strategies. Circulation, 144(9), 728-745.8. Twine, C. P., & McLain, A. D. (2010). Graft type for femoro-popliteal bypass sur-gery. Cochrane Database of Systematic Reviews, (5).9. Zhang, Y., Yu, Y., Akkouch, A., Dababneh, A., Dolati, F., & Ozbolat, I. T. (2015). Invitro study of directly bioprinted perfusable vasculature conduits. Biomaterials sci-ence, 3(1), 134-143.Hong, S., Kim, J. S., Jung, B., Won, C., & Hwang, C. (2019). Coaxial bioprinting ofcell-laden vascular constructs using a gelatin–tyramine bio-ink. Biomaterials sci-ence, 7(11), 4578-4587.Sasmal, P., Datta, P., Wu, Y. and Ozbolat, I.T., 2018. 3D bioprinting for modellingvasculature. Microphysiological systems, 2.Cui, H., Zhu, W., Huang, Y., Liu, C., Yu, Z. X., Nowicki, M., ... & Zhang, L. G. (2019).In vitro and in vivo evaluation of 3D bioprinted small-diameter vasculature with smoothmuscle and endothelium. Biofabrication, 12(1), 015004.Dahl, S. L., Kypson, A. P., Lawson, J. H., Blum, J. L., Strader, J. T., Li, Y., ... & Nikla-son, L. E. (2011). Readily available tissue-engineered vascular grafts. Science trans-lational medicine, 3(68), 68ra9-68ra9.Sasmal, P., Datta, P., Wu, Y. and Ozbolat, I.T., 2018. 3D bioprinting for modellingvasculature. Microphysiological systems, 2.Ott, H. C., & Mathisen, D. J. (2011). Bioartificial tissues and organs: are we ready totranslate?. Lancet (London, England), 378(9808), 1977-1978.Caliskan, E., De Souza, D. R., Boening, A., Liakopoulos, O. J., Choi, Y. H., Pepper,J., ... & Emmert, M. Y. (2020). Saphenous vein grafts in contemporary coronary arterybypass graft surgery. Nature Reviews Cardiology, 17(3), 155-169.Fan, L., Cao, M., Gao, S., Wang, T., Wu, H., Peng, M., ... & Nie, M. (2013). Prepara-tion and characterization of sodium alginate modified with collagen peptides. Carbo-hydrate polymers, 93(2), 380-385.Ullah, S., & Chen, X. (2020). Fabrication, applications and challenges of natural bio-materials in tissue engineering. Applied Materials Today, 20, 100656.Hu, T., & Lo, A. C. (2021). Collagen–Alginate Composite Hydrogel: Application in Tis-sue Engineering and Biomedical Sciences. Polymers, 13(11), 18Matai, I., Kaur, G., Seyedsalehi, A., McClinton, A., & Laurencin, C. T. (2020). Progressin 3D bioprinting technology for tissue / organ regenerative engineering. Biomateri-als, 226, 119536.Majesky, M. W. (2018). Vascular development. Arteriosclerosis, thrombosis, and vas-cular biology, 38(3), e17-e24.Kim, M. J., Park, P. J., Koo, B. H., Lee, S. G., Byun, G. Y., & Lee, S. R. (2020). Asso-ciation between venous reflux and diameter of great saphenous vein in lower thigh.Journal of Vascular Surgery: Venous and Lymphatic Disorders, 8(1), 100-105.Ketenciler, S., Boyacıoğlu, K., Akdemir, İ., Kömürcü, G., & Polat, A. (2018). Autolo-gous saphenous vein panel graft for vascular reconstruction. Annals of Vascular Sur-gery, 53, 117-122.Konig, G., McAllister, T. N., Dusserre, N., Garrido, S. A., Iyican, C., Marini, A., ... &L'Heureux, N. (2009). Mechanical properties of completely autologous human tissueengineered blood vessels compared to human saphenous vein and mammary artery.Biomaterials, 30(8), 1542-1550.Quint, C., Arief, M., Muto, A., Dardik, A., & Niklason, L. E. (2012). Allogeneic humantissue-engineered blood vessel. Journal of vascular surgery, 55(3), 790-798.Clements, R. H., & Palepu, R. (2007). In vivo comparison of the coagulation capabilityof SonoSurg and Harmonic Ace on 4 mm and 5 mm arteries. Surgical endos-copy, 21(12), 2203-2206.Hoganson, D. M., Cooper, D. A., Rich, K. N., Piekarski, B. L., Gui, L., Gaut, J. P., ...& Emani, S. M. (2018). Flow Preservation of Umbilical Vein for Autologous Shunt andCardiovascular Reconstruction. The Annals of thoracic surgery, 105(6), 1809-1818.Olsen, A. L., & Long, M. (2018). Comparison of catheter thrombogenicity in a modifiedchandler loop model using goat blood. Journal of Biomedical Materials Research PartA, 106(12), 3143-3151.Smethurst, P. A., Onley, D. J., Jarvis, G. E., O'Connor, M. N., Knight, C. G., Herr, A.B., ... & Farndale, R. W. (2007). Structural basis for the platelet-collagen interaction:the smallest motif within collagen that recognizes and activates platelet GlycoproteinVI contains two glycine-proline-hydroxyproline triplets. Journal of Biological Chemis-try, 282(2), 1296-1304.Westerhof, N., Boer, C., Lamberts, R. R., & Sipkema, P. (2006). Cross-talk betweencardiac muscle and coronary vasculature. Physiological reviews, 86(4), 1263-1308.Rusch, R., Trentmann, J., Hummitzsch, L., Rusch, M., Aludin, S., Haneya, A., ... &Berndt, R. (2019). Feasibility of a circulation model for the assessment of endovascu-lar recanalization procedures and periprocedural thromboembolism in-vitro. Scientificreports, 9(1), 1-6.Helms, F., Haverich, A., Böer, U., & Wilhelmi, M. (2021). Transluminal compressionincreases mechanical stability, stiffness and endothelialization capacity of fibrin-basedbioartificial blood vessels. Journal of the Mechanical Behavior of Biomedical Materi-als, 124, 104835.Zhang, Y., Yu, Y., Akkouch, A., Dababneh, A., Dolati, F., & Ozbolat, I. T. (2015). Invitro study of directly bioprinted perfusable vasculature conduits. Biomaterials sci-ence, 3(1), 134-143.Baudin, B., Bruneel, A., Bosselut, N., & Vaubourdolle, M. (2007). A protocol for isola-tion and culture of human umbilical vein endothelial cells. Nature protocols, 2(3), 481-485.Mead, L. E., Prater, D., Yoder, M. C., & Ingram, D. A. (2008). Isolation and character-ization of endothelial progenitor cells from human blood. Current protocols in stem cellbiology, 6(1), 2C-1.Emontzpohl, C., Simons, D., Kraemer, S., Goetzenich, A., Marx, G., Bernhagen, J., &Stoppe, C. (2017). Isolation of endothelial progenitor cells from healthy volunteers andtheir migratory potential influenced by serum samples after cardiac surgery. Journalof visualized experiments: JoVE, (120).Kong, L., Wang, Y., Wang, H., Pan, Q., Zuo, R., Bai, S., ... & Li, G. (2021). Conditionedmedia from endothelial progenitor cells cultured in simulated microgravity promoteangiogenesis and bone fracture healing. Stem cell research & therapy, 12(1), 1-14.Peters, E. B. (2018). Endothelial progenitor cells for the vascularization of engineeredtissues. Tissue Engineering Part B: Reviews, 24(1), 1-24.Jackson, R. J., Dao, M. L., & Lim, D. V. (1995). Modified FALGPA assay for cell-as-sociated collagenolytic activity. Journal of microbiological methods, 21(2), 209-215.Mørch, Ý. A., Donati, I., Strand, B. L., & Skjåk-Bræk, G. (2007). Molecular engineer-ing as an approach to design new functional properties of alginate. Biomacromole-cules, 8(9), 2809-2814.Xu, Y., Dai, J., Zhu, X., Cao, R., Song, N., Liu, M., ... & Yang, Y. (2021). BiomimeticTrachea Engineering via a Modular Ring Strategy Based on Bone-Marrow Stem Cellsand Atelocollagen for use in Extensive Tracheal Reconstruction. Advanced Materials,2106755.Tamaddon, M., Burrows, M., Ferreira, S. A., Dazzi, F., Apperley, J. F., Bradshaw, A.,... & Gentleman, E. (2017). Monomeric, porous type II collagen scaffolds promotechondrogenic differentiation of human bone marrow mesenchymal stem cells invitro. Scientific reports, 7(1), 1-10.Su, Y., Toftdal, M. S., Le Friec, A., Dong, M., Han, X., & Chen, M. (2021).3D Electro-spun Synthetic Extracellular Matrix for Tissue Regeneration. Small Science, 2100003.Aoun, S. G., El Ahmadieh, T. Y., El Tecle, N. E., Daou, M. R., Adel, J. G., Park, C. S.,& Bendok, B. R. (2015). A pilot study to assess the construct and face validity of theNorthwestern Objective Microanastomosis Assessment Tool. Journal of neurosur-gery, 123(1), 103-109.Movileanu, I., Harpa, M., Hussam, A.H., Harceaga, L., Chertes, A., Al Hussein, H.,Lutter, G., Puehler, T., Preda, T., Sircuta, C. and Cotoi, O., 2021. Pre-clinical Testingof Living Tissue Engineered Heart Valves for Pediatric Patients; Challenges and Op-portunities. Frontiers in cardiovascular medicine, 8, p.833.Nakajima, N., & Ikada, Y. (1995). Mechanism of amide formation by carbodiimide forbioconjugation in aqueous media. Bioconjugate chemistry, 6(1), 123-130.Hashimoto, T., Suzuki, Y., Tanihara, M., Kakimaru, K., Suzuki, K. (2004). Develop-ment of alginate wound dressings linked with hybrid peptides derived from lamininand elastin. Biomaterials, 25, 1407-1414.Crampton SP, Davis J, Hughes CC (2007). Isolation of human umbilical vein endo-thelial cells (HUVEC). J Vis Exp;(3):183.Fan, L., Cao, M., Gao, S., Wang, T., Wu, H., Peng, M. & Nie, M. (2013). Preparationand characterization of sodium alginate modified with collagen peptides. Carbohy-drate polymers, 93(2), 380-385.Simper, D., Stalboerger, P. G., Panetta, C. J., Wang, S., & Caplice, N. M. (2002).Smooth muscle progenitor cells in human blood. Circulation, 106(10), 1199-1204.Kolster, M., Wilhelmi, M., Schrimpf, C., Hilfiker, A., Ha-verich, A., & Aper, T. (2017).Outgrowing endothelial and smooth muscle cells for tissue engineering approaches.Journal of Tissue Engineering, 8, 2041731417698852.Mehta, M., Madl, C. M., Lee, S., Duda, G. N., & Mooney, D. J. (2015). The collagen Imimetic peptide DGEA enhances an osteogenic phenotype in mesenchymal stemcells when presented from cell-encapsulating hydrogels. Journal of Biomedical Mate-rials Research Part A, 103(11), 3516-3525.Phipps, M. C., Bonvallet, P., & Bellis, S. (2010). Enhancement of peptide coupling tohydroxyapatite and implant osseointegration through collagen mimetic peptide... Bio-materials, 31(36), 9586-9594.Zhang, W. M., Käpylä, J., Puranen, J. S., Knight, C. G., Tiger, C. F., Pentikäinen, O.T., ... & Gullberg, D. (2003). α11β1 integrin recognizes the GFOGER sequence ininterstitial collagens. Journal of Biological Chemistry, 278(9), 7270-7277.Castillo-Briceño, P., Bihan, D., Nilges, M., Hamaia, S., Meseguer, J., García-Ayala,A., & Mulero, V. (2011). A role for specific collagen motifs during wound healing andinflammatory response of fibroblasts in the teleost fish gilthead seabream. Molecularimmunology, 48(6-7), 826-834.Liao, H. J., Chen, H. T., & Chang, C. H. (2024). Peptides for Targeting Chondro-genic Induction and Cartilage Regeneration in Osteoarthritis. Cartilage,19476035241276406.

Claims

CLAIMS1. Bio-ink composition suitable for being printed into a bioartificial vascular graft, saidcomposition comprising:(a) a peptide-grafted alginate; and(b) smooth muscle cells, endothelial cells or cells which are capable of develop-ing into endothelial cells.

2. Bio-ink composition according to claim 1, wherein said alginate is sodium alginate.

3. Bio-ink composition according to claim 1 or 2, wherein the overall amount of peptidewhich is present in the peptide-grafted alginate is between 1% and 25% (w / w), andpreferably between 1% and 10% (w / w) based on the overall weight of the alginate.

4. Bio-ink composition according to any of claims 1-3, wherein said endothelial cells areHuman Umbilical Vein Endothelial Cells (HUVEC).

5. Bio-ink composition according to any of claims 1-3, wherein said cells which are ca-pable of developing into endothelial cells are Endothelial Progenitor Cells (EPC).

6. Bio-ink composition according to any of claims 1-5, wherein the composition com-prises 1x105 to 1x107 cells per ml.

7. Bio-ink composition according to any of claims 1-6, wherein the peptide has a size of3-50 amino acids.

8. Bio-ink composition according to any of claims 1-7, wherein the peptide in the peptide-grafted alginate comprises an amino acid sequence which is derived from or mimicsan extracellular matrix protein.

9. Bio-ink composition according to claims 8, wherein the peptide in the peptide-graftedalginate comprises an amino acid sequence which is derived from collagen, laminin,or elastin, and preferably human collagen, laminin, or elastin.

10. Bio-ink composition according to any of claims 1-9, wherein(a) the peptide in the peptide-grafted alginate comprises amino acid sequencesfrom more than one extracellular matrix protein, and / or(b) the alginate is grafted to two or more different peptides.

11. Bio-ink composition according to claim 9, wherein the peptide in the peptide-graftedalginate comprises or consists of a collagen-derived amino acid sequence.

12. Bio-ink composition according to claim 11, wherein the peptide in the peptide-graftedalginate comprises or consists of the sequence Leu-Gly-Pro-Ala.

13. Bio-ink composition according to claim 11, wherein the peptide in the peptide-graftedalginate comprises or consists of a sequence selected from SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:9 or SEQ ID NO:10.

14. Bio-ink composition according to any of claims 1-13, wherein the cells are allogenicor autologous in relation to the subject that shall receive the bioartificial vascular graft.

15. Method for manufacturing a bioartificial vascular graft, comprising(a) loading the bio-ink composition according to any of claims 1-14 into a 3Dprinter which is capable of printing viscous hydrogels;(b) contacting the bio-ink composition with a crosslinking solution comprising adivalent and / or trivalent cation in the 3D printer to induce crosslinking of thealginate; (c) printing the bio-ink to obtain the bioartificial vascular graft.

16. Method according to claim 15, wherein the 3D printer comprises a print head with afirst chamber containing the bio-ink composition and a second chamber containingthe crosslinking solution, and wherein the fluids of the first and second chamber aremixed upon printing.

17. Method according to claim 15 or 16, wherein the crosslinking solution used in step (b)comprises Ca2+ ions.

18. Method according to any of claims 15-17, further comprising a step (d) in which thebioartificial vascular graft obtained from step (c) is transferred into a solution compris-ing a divalent and / or trivalent cation for further crosslinking.

19. Method according to any of claims 15-18, wherein the bioartificial vascular graft ob-tained from step (c) or step (d) is transferred in a bioreactor for cell culturing.

20. Use of a bio-ink composition according to any of claims 1-14 for manufacturing a bio-artificial vascular graft.

21. Bioartificial vascular graft obtainable by the method of any of claims 15-19.

22. Bioartificial vascular graft according to claim 21 for use in medicine.

23. Bioartificial vascular graft according to claim 21 for use in vascular or cardiovasculartherapy, and preferably in vessel repair by or pass surgery.

24. Use of a vascular graft according to claim 21 for in vitro drug screenings or as an in-vitro training model for endovascular or surgical training.

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