Synthetic bio-ink formulations

Abstract

A heterogeneous component free synthetic bio-ink formulation comprising a polymer derived from poly (asparagine) (PASPAm) for 3D bioprinting applications, a method of producing a three-dimensional object using the synthetic bio-ink formulation, and a three-dimensional object obtainable by the method are provided.

Description

[0001] SEQUENCE LISTING

[0002] This application claims priority benefit of U.S. Provisional Patent Application No. 63 / 582,304, filed September 13, 2023, the entire contents of which are incorporated herein in their entirety by this reference.

[0003] The instant application contains a Sequence Listing which has been filed in XML format and which is incorporated by reference herein in its entirety. The XML copy, created on July 25, 2024, is named P23-105-WO-PCT_SL.xml and is 11,772 bytes in size. TECHNICAL FIELD

[0004] The present invention provides xeno-free synthetic bioink formulations for 3D bioprinting applications comprising polymeric scaffolds based on poly(aspartamide) (PAspAm). The bioink formulations of the present invention are biocompatible, biodegradable, and promote cell functionality. Furthermore, they exhibit better printability and cross-linkability to ensure high dimensional fidelity and structural integrity of the printed structures made therefrom. In addition, the bioink formulations of the present invention can be tuned to exhibit beneficial shear-thinning properties.

[0005] Further provided is a method of producing a three-dimensional object, wherein the bioink formulation is applied to a 3D bioprinting technique to produce the three-dimensional object. In addition, a three-dimensional object obtainable by the method is provided. The three-dimensional object can be used for tissue engineering, regenerative medicine, cell delivery, drug delivery, drug development, wound dressing, biosensors, cosmetics, hygiene products, medical devices, implantable electronics, and / or other biomedical applications. BACKGROUND

[0006] Biofabrication is an interdisciplinary field that combines principles of engineering, biology, and materials science and has the potential to generate constructs that highly mimic the composition and hierarchical structure of natural tissues. Such constructs can be applied to various biomedical uses, including tissue engineering, regenerative medicine, and drug development.

[0007] Among several manufacturing methods, 3D bioprinting offers great potential in the manufacturing of functional tissues as it provides better control over the spatial arrangement of the matrix, the construct fidelity, and the composition reproducibility of the constructs. 3D bioprinting technology enables the precise placement of cells at spatially predetermined locations within a defined 3D structure.

[0008] The most common 3D bioprinting technologies include DLP-based bioprinting, droplet-based bioprinting, extrusion-based bioprinting, forward transfer bioprinting, inkjet bioprinting, integrated bioprinting, laser-induced bioprinting, stereolithography-based bioprinting, magnetic bioprinting, and volumetric bioprinting. These 3D bioprinting technologies enable precise placement / positioning within a predetermined spatial location within a defined 3D structure.

[0009] One of the key components of 3D bioprinting is bioink, used to form tissue constructs. Bioink is an aqueous solution of one or more biomaterials in the form of a hydrogel, optionally containing one or more desired cell types. Therefore, bioink should be able to deliver cells during the printing process and support cell growth after printing. To this end, bioink should meet certain material and biological requirements.

[0010] Typical material properties include printability, crosslinkability, matrix stiffness, shear stress protection during printing, and dimensional fidelity of the printed structure during subsequent cell culture.

[0011] Biological requirements mainly include degradability, cell compatibility, and promotion of cell bioactivity in subsequent cell culture.

[0012] Among various biomaterials, hydrophilic polymers are the most prominent materials used in bioink formulations. To date, natural polymers, such as collagen, gelatin, hyaluronic acid, alginate, and chitosan, have played a central role in bioink formulations due to their availability and ability to provide adaptable scaffold systems for the structural and functional organization of cells.

[0013] Natural polymers possess unique properties, such as biocompatibility and biodegradability, making them well-suited for many biomedical applications. However, natural polymers also have several inherent drawbacks, including the high structural variability of polymers derived from different animals and poor batch-to-batch consistency in manufacturing processes. Natural polymers are also structurally more complex and difficult to chemically modify to achieve the desired rheological and mechanical properties. Furthermore, natural polymers pose a high risk of contamination from exogenous factors. Additionally, the use of animal-derived polymers raises regulatory and ethical concerns. Therefore, there is a need for materials derived from non-exogenous components, such as bio-ink formulations using synthetic polymers.

[0014] Bioinks derived from synthetic polymers can readily overcome some of the inherent challenges of bioinks based on natural polymers. For example, synthetic polymers have well-defined, less complex structures that can be readily modified using several functionalization strategies to provide greater control over cell-mediated degradation and cell functions, including adhesion, proliferation, and morphogenesis. Furthermore, synthetic polymers can be manufactured on a large scale with the high batch-to-batch consistency required for reproducible results.

[0015] KR 20180025117 A relates to a bio-ink containing a polyester-based copolymer with a controlled melting point, a biocompatible structure containing the same, and a method for manufacturing the same. The polyester-based copolymer is synthesized using at least one alcohol-based initiator selected from diethylene glycol ethyl ether, polypropylene glycol, and methoxy (ethylene glycol).

[0016] KR 20200017606 A relates to scaffolds for tissue regeneration, methods for manufacturing the same, and bio-ink materials for 3D printing using the same. The bio-ink material for 3D printing is based on physically cross-linked casein-polyvinyl alcohol hydrogels.

[0017] WO 2019 / 236891 A1 relates to pharmaceutical bio-inks, pharmaceutical formulations, and methods for printing pharmaceutical bio-inks. The pharmaceutical bio-inks comprise a hydrophobic photocurable resin capable of curing by visible light, wherein, in particular, poly(ethylene glycol) diacrylate (PEGDA) is used as such a hydrophobic photocurable resin.

[0018] WO 2021 / 234141 A1 relates to a hydrogel formed by combining polyethylene glycol (PEG) with heparin and positively charged immune molecules.

[0019] Many of these bio-ink formulations made from synthetic polymers contain non-degradable main-chain structures and may hinder cell function. Furthermore, they lack structural units that promote cell functions such as adhesion, proliferation, and morphogenesis.

[0020] Therefore, there remains a continued need to develop improved bio-ink formulations suitable for 3D bioprinting applications that overcome the known shortcomings of existing technologies. In particular, there is a need for improved bio-ink formulations suitable for 3D bioprinting that are free of foreign components, biocompatible, biodegradable, and promote cell function, exhibit better printability and crosslinkability to ensure high dimensional fidelity and structural integrity of the printed structures made from them, and can be tuned to exhibit beneficial shear-thinning properties.

[0021] Purpose of the invention

[0022] This invention aims to overcome the drawbacks of existing bioinks, including those derived from natural and synthetic polymers. These drawbacks include, in particular, batch-to-batch inconsistencies, difficulty in fine-tuning polymer properties, scaling up manufacturing, the risk of exogenous contamination, the non-degradability of synthetic polymers, and the lack of structures that promote cell function.

[0023] Therefore, the main objective of this invention is to provide a bio-ink formulation suitable for 3D bioprinting.

[0024] Another object of the present invention is to provide a bio-ink formulation that is free of foreign ingredients (i.e., free of one or more animal-derived components).

[0025] Another object of the present invention is to provide a biocompatible, biodegradable bioink formulation that does not exhibit cytotoxicity.

[0026] Another object of the present invention is to provide a bio-ink formulation that maintains high cell viability and promotes cell function, including but not limited to attachment, proliferation, metabolism, differentiation and extracellular matrix deposition.

[0027] Another object of the present invention is to provide a bio-ink formulation that exhibits better printability and crosslinkability to ensure high dimensional fidelity and structural integrity of the printed structures made therefrom, thereby maintaining the 3D bioprinted structures.

[0028] Another object of the present invention is to provide a bio-ink formulation that exhibits beneficial shear-thinning properties. Summary of the Invention

[0029] The embodiments of the present invention described below provide technical solutions to the above-mentioned objectives. In a first embodiment, the present invention provides a bio-ink formulation comprising a polymer, wherein the polymer comprises repeating unit U1 and repeating unit U1': , in: R 1 Selected from alkylene groups, preferably from C1-C64 groups. 20 Alkylene, more preferably selected from C1-C 10 Alkylene, most preferably selected from methylene, ethylene, and propylene; R 11 Selected from hydrogen or alkyl groups, preferably selected from hydrogen or C1-C 20 Alkyl groups, more preferably selected from hydrogen or C1-C 10 Alkyl groups, most preferably selected from hydrogen, methyl, ethyl, and propyl; A 1 It is the structural part that imparts hydrophilicity to the polymer; n1 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; and m1 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000.

[0030] In a second embodiment, the present invention provides a method for producing three-dimensional objects, wherein a bio-ink formulation according to a first embodiment of the present invention is applied to 3D bioprinting technology to produce the three-dimensional objects.

[0031] In a third embodiment, the present invention provides a three-dimensional object that can be obtained or acquired by a method for producing a three-dimensional object according to a second embodiment of the present invention.

[0032] The invention is further described in the embodiments detailed below. Attached Figure Description

[0033] Figure 1 A) Schematic synthesis of PAspAm-MA (1); B) PAspAm-MA (1) 1 H NMR spectrum.

[0034] Figure 2 Rheological analysis of time-dependent shear storage modulus (G') and shear loss modulus (G'') of PAspAm-MA (1) irradiated with visible light (405 nm) at room temperature and concentrations of 5% and 10% wt / v.

[0035] Figure 3 Cell viability in PAspAm-MA (1) hydrogels; A) Live / dead stained 3T3 fibroblasts encapsulated in PAspAm-MA (1) hydrogels after 1 and 3 days of culture, scale bar = 300 µm; B) Cell viability calculated as the percentage of live cells from live / dead stained images; C) Cell metabolism as measured by PrestoBlue® fluorescence assay. p < 0.05.

[0036] Figure 4 PAspAm-MA (1) bio-ink is printable via digital light processing (DLP) with a print size of 25mm. 13mm M-shape, 0.6mm.

[0037] Figure 5 A) Schematic synthesis of PAspAm-Tyr (2); B) PAspAm-Tyr (2) 1 H NMR spectrum.

[0038] Figure 6 Rheological analysis of the time-dependent shear storage modulus (G') of PAspAm-Tyr (2) irradiated with visible light (405 nm).

[0039] Figure 7 PAspAm-Tyr (2) Printability of bio-ink via digital light processing (DLP); A) Size of 9 mm 9 mm A) 0.5 mm grid shape; B) Print size 34 mm 7.2 mm The word "BIOINK" is 0.5mm thick.

[0040] Figure 8 Cell growth (hMSCs) in DLP-printed PAspAm-Tyr (2) bio-ink hydrogels; A) Live / dead stained hMSCs encapsulated in bioprinted PAspAm-Tyr (2) hydrogels after 1, 7, and 14 days of culture. Scale bar = 750 µm; B) Cell metabolism as measured by PrestoBlue® fluorescence assay. p<0.05. C) Cell viability is calculated as the percentage of live cells (gray) from live / dead stained images.

[0041] Figure 9 Cell growth (3T3 fibroblasts) in DLP-printed PAspAm-Tyr-RGD+ (3) and PAspAm-Tyr (2) bio-ink hydrogels; PEGDA (6k) was used as a control; live / dead stained 3T3 fibroblasts encapsulated in bioprinted PAspAm-Tyr-RGD+ (3), PAspAm-Tyr (2) and PEGDA (6k) hydrogels after 1, 7, 14 and 21 days of culture; scale bar = 750 µm.

[0042] Figure 10 Cell growth (3T3 fibroblasts) in DLP-printed PAspAm-Tyr-RGD+ (3) and PAspAm-Tyr (2) bio-ink hydrogels; PEGDA (6kJ) was used as a control group; A) Cell metabolism as measured by PrestoBlue® fluorescence assay. p<0.05; B) Cell viability calculated as the percentage of live cells (gray) from live / dead stained images.

[0043] Figure 11 A) Schematic synthesis of PAspAm-Nor (4); B) PAspAm-Nor (4)1 H NMR spectrum.

[0044] Figure 12 Crosslinking mechanism of PAspAm-Nor-RGD++ (4a) bioink based on thiol-ene click chemistry.

[0045] Figure 13 Rheological analysis of time-dependent shear storage modulus (G') of PAspAm-Nor (4) and PAsp-Am-Nor-RGD++ (4a) bioinks irradiated with visible light (405 nm).

[0046] Figure 14 Cell growth (3T3 fibroblasts) in DLP-printed PAspAm-Nor (4) and PAspAm-Nor-RGD++ (4a) bio-ink hydrogels; A) Live / dead stained 3T3 fibroblasts encapsulated in bioprinted PAspAm-Nor (4) and PAspAm-Nor-RGD++ (4a) hydrogels after 1, 7, 14, 21 and 28 days of culture, scale bar = 1500 µm; B) Cell metabolism as measured by PrestoBlue® fluorescence assay. Detailed Implementation

[0047] In a first embodiment of the present invention, a bio-ink formulation comprising a polymer is provided, wherein the polymer comprises repeating unit U1 and repeating unit U1': , in: R 1 Selected from alkylene groups, preferably from C1-C64 groups. 20 Alkylene, more preferably selected from C1-C 10 Alkylene, most preferably selected from methylene, ethylene, and propylene; R 11 Selected from hydrogen or alkyl groups, preferably selected from hydrogen or C1-C 20 Alkyl groups, more preferably selected from hydrogen or C1-C 10 Alkyl groups, most preferably selected from hydrogen, methyl, ethyl, and propyl; A 1 It is the structural part that imparts hydrophilicity to the polymer; n1 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; and m1 is an integer from 1 to 5000, preferably from 10 to 5000, and more preferably from 100 to 5000.

[0048] A in the repeating units U1 and U1' of the polymer1 This refers to the structural portion that imparts hydrophilicity to the polymer. The structural portion that imparts hydrophilicity to the polymer refers to a structural portion containing at least one polar and / or ionic group capable of interacting with water through the formation of secondary bonds (e.g., hydrogen bonds), dipole bonds, electrostatic interactions, and / or van der Waals interactions with water molecules.

[0049] Preferably, A in the repeating units U1 and U1' of the polymer 1 It contains one or more groups selected from -OH, -SH, -NH2, -CO-NH2 and -CO2H.

[0050] Preferably, A in the repeating units U1 and U1' of the polymer 1 It includes water-soluble structural portions. Such water-soluble structural portions include water-soluble polymer structural portions, such as, but not limited to, polyethylene oxide, polyvinyl alcohol, and polyacrylamide structural portions.

[0051] Preferably, A in the repeating units U1 and U1' of the polymer 1 Contains an alkylene unit -(CH2) x - and / or ethylene oxide units -(CH2-CH2-O) y - where x and y are independent of each other and are 1 to 2,000, preferably 1 to 100, more preferably integers from 1 to 10, and most preferably 1.

[0052] More preferably, A in the repeating units U1 and U1' of the polymer 1 It contains one or more groups selected from -OH, -SH, -NH2, -CO-NH2, and -CO2H; and an alkylene unit -(CH2). x - and / or ethylene oxide units -(CH2-CH2-O) y - where x and y are independent of each other and are 1 to 2,000, preferably 1 to 100, more preferably integers from 1 to 10, and most preferably 1.

[0053] Most preferably, A in the repeating units U1 and U1' of the polymer 1Selected from -CH2-CH2-OH, -CH2-CH2-O-CH2-CH2-OH, -CH2-CH2-O-CH2-CH2-O-CH2-CH2-OH, -CH2-CH2-O-CH2-CH2-OCH3, -CH2-CH2-O-CH2-CH2-O-CH2-CH2-OCH3, -CH2-CH2-S H, -CH2-CH2-O-CH2-CH2-SH, -CH2-CH2-NH2, -CH2-CH2-O-CH2-CH2-NH2, -CH2-CH2-CO2H, -CH2-CH2-O-CH2-CH2-CO2H, -CH2-CH2-CO2H, and -CH2-CH2-O-CH2-CH2-CO2H.

[0054] In a preferred embodiment of the invention, the polymer further comprises repeating unit U2 and repeating unit U2': , in: R 2 The components are independently selected from alkylene groups, preferably from C1-C2 groups. 20 Alkylene, more preferably selected from C1-C 10 Alkylene, most preferably selected from methylene, ethylene, and propylene; R 22 The components are independently selected from hydrogen or alkyl groups, preferably from hydrogen or C1-C groups. 20 Alkyl groups, more preferably selected from hydrogen or C1-C 10 Alkyl groups, most preferably selected from hydrogen, methyl, ethyl, and propyl; A 2 It is a structural part containing at least one reactive group that can further react to crosslink two or more polymer chains; n2 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; and m2 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000.

[0055] Preferably, A in repeating units U2 and U2' 2 The reactive groups contained therein are selected from groups that can undergo polymerization crosslinking reactions, groups that can undergo Michael crosslinking reactions or click chemistry-type crosslinking reactions, groups that can undergo oxidative crosslinking reactions, and groups that can undergo redox crosslinking reactions.

[0056] More preferably, A in the repeating units U2 and U2' of the polymer 2The reactive groups contained herein are selected from groups capable of undergoing polymerization and crosslinking reactions, wherein preferably the groups contain one or more vinyl or allyl groups.

[0057] More preferably, A in the repeating units U2 and U2' of the polymer 2 The reactive groups contained herein are selected from groups capable of undergoing Michael-type or click-chemical crosslinking reactions, wherein preferably the groups contain one or more acrylate, methacrylate, n-alkyne, cycloalkyne, amine, azide, carboxyl, hydrazine, hydroxyl, maleimide, norbornene, tetraazine, and / or thiol groups. Most preferably, A in the repeating units U2 and U2' of the polymer 2 Selected from the tyramine and tyrine structural moieties.

[0058] More preferably, A in the repeating units U2 and U2' of the polymer 2 The reactive groups contained therein are selected from groups capable of undergoing oxidative crosslinking reactions, wherein preferably the groups contain one or more aromatic structural moieties having one or more aromatic hydroxyl groups, wherein more preferably the groups contain one or more monophenol, diphenol, triphenol, oligophenol and / or polyphenol structural moieties, wherein particularly preferably the groups contain monophenol structural moieties. Most preferably, A in the repeating units U2 and U2' of the polymer 2 Selected from -CH2-CH2-C6H4-OH and -CH(CO2H)-CH2-C6H4-OH.

[0059] More preferably, A in the repeating units U2 and U2' of the polymer 2 The reactive groups contained therein are selected from groups capable of undergoing redox crosslinking reactions, wherein preferably the groups contain one or more thiol and / or amine groups. Most preferably, A in the repeating units U2 and U2' of the polymer 2 Selected from the glutathione and histidine structural moieties.

[0060] In a preferred embodiment of the invention, the bio-ink formulation comprises components capable of reacting with A 2 A reactive peptide compound. Preferably, the reactive peptide compound comprises one or more compounds capable of reacting with A. 2 Functional groups that react with the reactive groups contained therein. Preferably, they are functional groups capable of reacting with A. 2 The reactive functional groups contained therein are selected from allyl, styryl, vinyl, acrylate, methacrylate, maleimide and thiol.

[0061] Preferred reactive peptide compounds are RGD-containing peptide compounds having one or more functional groups selected from allyl, phenethyl, vinyl, acrylate, methacrylate, maleimide, and thiol. Particularly preferred reactive peptide compounds are H-Gly-Arg-Gly-Asp-Ser-Pro-Cys-OH (SEQ ID NO: 11) having a thiol group.

[0062] In a more preferred embodiment of the invention, the polymer further comprises (i) repeating unit U3 and repeating unit U3'; or (ii) repeating unit U4 and repeating unit U4'; or (iii) repeating unit U3, repeating unit U3', repeating unit U4 and repeating unit U4': , in: R 3 and R 4 The components are independently selected from alkylene groups, preferably from C1-C2 groups. 20 Alkylene, more preferably selected from C1-C 10 Alkylene, most preferably selected from methylene, ethylene, and propylene; R 33 and R 44 The components are independently selected from hydrogen or alkyl groups, preferably from hydrogen or C1-C groups. 20 Alkyl groups, more preferably selected from hydrogen or C1-C 10 Alkyl groups, most preferably selected from hydrogen, methyl, ethyl, and propyl; A 3 It is the structural part that gives the polymer cells their function; A 4 It is the structural part that imparts shear-thinning properties to the polymer; In case (i): n3 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; m3 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; and The sum of n1, n2, n3, m1, m2, and m3 is greater than 5, preferably greater than 50, and more preferably greater than 500; or In case (ii): n4 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; m4 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; and The sum of n1, n2, n4, m1, m2, and m4 is greater than 5, preferably greater than 50, more preferably greater than 500, or In case (iii): n3 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; m3 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; n4 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; m4 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; and In case (iii), the sum of n1, n2, n3, n4, m1, m2, m3 and m4 is greater than 7, preferably greater than 50, and more preferably greater than 500.

[0063] The A in the repeating units U3 and U3' of the polymer 3 It is the structural part that endows the polymer with cellular functions. Preferred cellular functions include, but are not limited to, attachment, proliferation, metabolism, differentiation, and extracellular matrix deposition.

[0064] Preferably, A in the repeating units U3 and U3' of the polymer 3 It contains one or more synthetic functional peptide sequences present in natural proteins, wherein the natural proteins are preferably fibronectin, laminin, collagen, elastin, albumin, immunoglobulin or silk.

[0065] Preferred synthetic functional peptide sequences are selected from RGD, YIGSR (SEQ ID NO: 1), IKVAV (SEQ ID NO: 2), DGEA (SEQ ID NO: 3), PHSRN (SEQ ID NO: 4), PRARI (SEQ ID NO: 5), and (GAGAGS). p (SEQ ID NO: 6), (VPGXG) n (SEQ ID NO: 7), (GPO) m And Gly-Lys-Gly-Tyr-Gly-Arg-Gly-Asp-Ser-Pro-Gly (SEQ ID NO: 8), where p, n and m are mutually independent integers ≥ 1.

[0066] The repeating units U4 and U4' of the polymer contain A 4 It is the structural part that imparts the shear-thinning properties to the polymer.

[0067] Preferably, A in the repeating units U4 and U4' of the polymer 4 It is the structural part that acquires shear-thinning properties due to supramolecular interactions.

[0068] Preferred supramolecular interactions are host-guest or self-assembly interactions, wherein such interactions preferably include interactions between cyclodextrin, adamantane, cucurbita[8]urea, cholesterol, polyethylene glycol and ureidopyrimidinone.

[0069] In a preferred embodiment of the invention, A in the repeating units U4 and U4' of the polymer 4 It contains one or more cyclodextrin, adamantane, cucurbita[8]urea, cholesterol, polyethylene glycol or ureidopyrimidinone structural moieties.

[0070] In a more preferred embodiment of the invention, A in the repeating units U4 and U4' of the polymer 4 It is a structural part of cyclodextrin, adamantane, cucurbita[8]urea, cholesterol, polyethylene glycol or ureidopyrimidinone.

[0071] In a particularly preferred embodiment of the invention, the polymer is represented by Formula I:

[0072] Formula I

[0073] in: R 1 R 2 R 3 and R 4 The components are independently selected from alkylene groups, preferably from C1-C2 groups. 20 Alkylene, more preferably selected from C1-C 10 Alkylene, most preferably selected from methylene, ethylene, and propylene; R 11 R 22 R 33 and R 44 The components are independently selected from hydrogen or alkyl groups, preferably from hydrogen or C1-C groups. 20 Alkyl groups, more preferably selected from hydrogen or C1-C 10 Alkyl groups, most preferably selected from hydrogen, methyl, ethyl, and propyl; A 1 It is the structural part that imparts hydrophilicity to the polymer; A 2 It is a structural part containing at least one reactive group that can further react to crosslink two or more polymer chains; A 3 It is the structural part that gives the polymer cells their function; A4 It is the structural part that imparts shear-thinning properties to the polymer; n1 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; m1 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; n2 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; m2 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; n3 is an integer from 0 to 5,000, preferably from 1 to 5,000, more preferably from 10 to 5,000, and most preferably from 100 to 5,000; m3 is an integer from 0 to 5,000, preferably from 1 to 5,000, more preferably from 10 to 5,000, and most preferably from 100 to 5,000; n4 is an integer from 0 to 5,000, preferably from 1 to 5,000, more preferably from 10 to 5,000, and most preferably from 100 to 5,000; and m4 is an integer from 0 to 5000, preferably from 1 to 5000, more preferably from 10 to 5000, and most preferably from 100 to 5000; The sum of n1, n2, n3, n4, m1, m2, m3 and m4 is greater than 5, preferably greater than 7, more preferably greater than 50, and most preferably greater than 500.

[0074] Repeating units U1, U1', U2, U2', U3, U3', U4, and U4' can be distributed in any order in the polymer shown in Formula I. The polymer can therefore exist, for example, as a random copolymer, as an alternating copolymer, and / or as a block copolymer. (Astro) The connection of the two parts of the polymer shown in Equation I.

[0075] Structural part A in the polymer shown in Formula I 1 To A 4 The preferred implementation scheme is shown above.

[0076] In a preferred embodiment of the invention, the polymer in the bio-ink formulation has a weight-average molecular weight (Mn) in the range of 1,000 Da (1 kDa) to 100,000,000 Da (100 MDa), preferably 10,000 Da (10 kDa) to 10,000,000 Da (10 MDa), more preferably 20,000 Da (20 kDa) to 5,000,000 Da (5 MDa), and most preferably 30,000 Da (30 kDa) to 1,000,000 Da (1 MDa). w Weight-average molecular weight (M) w It can be determined by any standard method known to those skilled in the art, such as GPC.

[0077] In a preferred embodiment of the invention, the mass concentration of the polymer in the bio-ink formulation is from 0.1% (wt / v) to 99% (wt / v), preferably from 1% (wt / v) to 70% (wt / v), more preferably from 2% (wt / v) to 40% (wt / v), particularly preferably from 5% (wt / v) to 30% (wt / v), and most preferably from 5% (wt / v) to 20% (wt / v). Depending on the type of 3D bioprinting technology applied, those skilled in the art can determine the appropriate mass concentration of the polymer in the bio-ink formulation.

[0078] In a preferred embodiment of the invention, the bioink formulation comprises one or more diluents. Such diluents typically act as solvents and / or dispersants to dilute the polymer in the bioink formulation. Preferred diluents are compounds that are liquid at room temperature. Preferred diluents are water, cell culture media such as DMEM, RPMI, and MEM, or water-based buffer systems such as phosphate-buffered saline (PBS), Hank's buffer, Earle's balanced salt solution, Tyrode buffer, HEPES buffer, etc.

[0079] In a preferred embodiment of the invention, the bioink formulation further comprises one or more selected from crosslinking agents and crosslinking additives. The crosslinking agent serves to crosslink the polymer in the bioink formulation to form a crosslinked three-dimensional hydrogel. The crosslinking additive serves to react with the polymer in the bioink formulation to form a crosslinked three-dimensional hydrogel.

[0080] Preferred crosslinking agents are selected from photoinitiators, thermal initiators, basic initiators, oxidative initiators, enzymes that mediate oxidative crosslinking, and reagents that mediate redox crosslinking.

[0081] Preferred photoinitiators are selected from acetophenone, p-anisoloyl, benzoyl, benzoin, benzophenone, 2-benzoylbenzoic acid, 4,4'-bis(diethylamino)benzophenone, 4,4'-bis(dimethylamino)benzophenone, benzoin methyl ether, benzoin isopropyl ether, benzoin isobutyl ether, benzoin ethyl ether, 4-benzoylbenzoic acid, 2,2'-bis(2-chlorophenyl)-4,4',5,5' -Tetraphenyl-1,2'-Bimidazole, Methyl 2-benzoylbenzoate, 2-(1,3-benzo[m]dioxacyclopenten-5-yl]-4,6-bis(trichloromethyl)-1,3,5-triazine, 2-benzyl-2-(dimethylamino)-4'-morpholinophenylbutanone, (±)-camphorquinone, 2-chlorothiazolone, 4,4'-dichlorobenzophenone, 2,2-diethoxyacetophenone, 2,2-dimethoxy 2-phenylacetophenone, 2,4-diethylthioxanthion-9-one, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, 1,4-dibenzoylbenzene, eosin Y, 2-ethylanthraquinone, 1-hydroxycyclohexylphenyl ketone, 2-hydroxy-2-methylphenylacetone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, 2-isopropylthioxanthionone, phenyl(2,4,6-trimethyl)phosphine oxide Lithium benzoylphosphine, 2-methyl-4'-(methylthio)-2-morpholinophenylacetone, 2-nitrosophenylacetone, 2-phenyl-2-(p-toluenesulfonyloxy)acetophenone, phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide, tris(2,2'-bipyridyl)ruthenium(II) hexahydrate, and combinations of tris(2,2'-bipyridyl)ruthenium(II) hexahydrate and sodium persulfate.

[0082] More preferred photoinitiators are selected from eosin Y, 2-hydroxy-4'(2-hydroxyethoxy)-2-methylphenylacetone, phenyl-2,4,6-trimethylbenzoyl lithium phosphine, tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate, and combinations of tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate and sodium persulfate.

[0083] Preferred thermal initiators are selected from tert-amyl peroxybenzoate, 4,4-azobis(4-cyanopentanoic acid), 1,1'-azobis(cyclohexanecarboxynitrile), 2,2'-azobisisobutyronitrile (AIBN), 2,2'-azobis(2-methylpropanediamine) dihydrochloride, benzoyl peroxide, 2,2-bis(tert-butylperoxy)butane, 1,1-bis(tert-butylperoxy)cyclohexane, 2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, and 2,5-bis(tert-butylperoxy) -2,5-Dimethyl-3-hexyne, bis(1-(tert-butylperoxy)-1-methylethyl)benzene, 1,1-bis(tert-butylperoxy)-3,3,5-trimethylcyclohexane, tert-butyl hydroperoxide, tert-butyl peracetate, tert-butyl peroxide, tert-butyl peroxybenzoate, tert-butyl peroxyisopropyl carbonate, cumene hydroperoxide, cyclohexanone peroxide, dicumyl peroxide, lauroyl peroxide, 2,4-pentanedione peroxide, peracetic acid, ammonium persulfate, potassium persulfate, and sodium persulfate.

[0084] More preferred thermal initiators are selected from 4,4-azobis(4-cyanopentanoic acid), 2,2'-azobisisobutyronitrile (AIBN), 2,2'-azobis(2-methylpropanediamine) dihydrochloride, benzoyl peroxide, tert-butyl peroxide, ammonium persulfate, potassium persulfate, and sodium persulfate.

[0085] Preferred basic initiators are selected from hydroxides, amines, and amides. More preferred basic initiators are selected from MOH, MOR, NH3, RNH2, and R2NM, wherein M is an alkali metal, preferably Li, Na, or K; and R is a carbyl moiety, preferably C1-C6 alkyl. The most preferred basic initiator is selected from OH. – NH3 and ( i Pr)2NLi(LDA).

[0086] Preferred oxidation initiators are selected from hydroxyl (H) ) and peroxy group (RO) ), peroxynitrite / peroxynitroso (ONOOH / ONOO) – ), nitrogen dioxide (NO2) ), nitrosopyrocarbonate (ONOOCO2) – ), carbonate (CO3) – R is a divalent carbon moiety, preferably C1-C6 alkyl, and L is a lipid moiety.

[0087] The preferred enzymes mediating oxidative cross-linking are selected from the peroxidase family, including horseradish peroxidase, myeloperoxidase, laccase, etc.

[0088] Preferred reagents for mediating redox crosslinking are selected from peroxides, hypochlorous acid, chloramine, hypobromic acid, bromamine, hypothiocyanate, nitryl groups, peroxynitrite, and other nitrates.

[0089] In a preferred embodiment of the present invention, the bio-ink formulation comprises two crosslinking agents selected from the above list.

[0090] In a more preferred embodiment of the invention, the bio-ink formulation comprises lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid.

[0091] In a more preferred embodiment of the invention, the bio-ink formulation comprises a combination of tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate and sodium persulfate.

[0092] Preferred crosslinking additives are compounds comprising two or more polymerizable groups having at least one C=C double bond. Preferred polymerizable groups having at least one C=C double bond are selected from acrylates, methacrylates, allyl, styrene, and vinyl groups. Alternatively, preferred crosslinking additives are compounds comprising two or more thiol groups, preferably three or more thiol groups, more preferably four thiol groups. Examples include 4-arm-PEG-SH.

[0093] In a preferred embodiment of the invention, the concentration of the crosslinking agent in the bio-ink formulation is 0.1 mM to 100 mM, preferably 0.2 mM to 50 mM, more preferably 0.5 mM to 40 mM, particularly preferably 1 mM to 30 mM, even more preferably 5 mM to 25 mM, and most preferably 15 mM to 25 mM.

[0094] The bio-ink formulation of the present invention can be used in different 3D bioprinting technologies, including DLP-based bioprinting, droplet-based bioprinting, extrusion-based bioprinting, forward transfer bioprinting, inkjet bioprinting, integrated bioprinting, laser-induced bioprinting, stereolithography-based bioprinting, magnetic bioprinting, and volumetric bioprinting.

[0095] Furthermore, the bio-ink formulation of this invention is compatible with several human and non-human cell types, and therefore can be used to print various tissue constructs. This is illustrated by the cell lines used in the experimental examples below, such as mouse 3T3 fibroblasts, human bone marrow-derived mesenchymal stem cells, etc., which should not be construed as limiting in any way.

[0096] In summary, the bio-ink formulations described herein overcome the known drawbacks of the prior art. Specifically, the bio-ink formulations described herein are suitable for 3D bioprinting; they are biocompatible, biodegradable, and do not exhibit cytotoxicity. They maintain high cell viability and promote cell function, including but not limited to attachment, proliferation, metabolism, differentiation, and extracellular matrix deposition. They exhibit better printability and crosslinkability to ensure high dimensional fidelity and structural integrity of the printed structures made from them, thus preserving the 3D bioprinted structures. Furthermore, they exhibit beneficial shear-thinning properties.

[0097] In a second embodiment of the invention, a method for producing a three-dimensional object is provided, wherein a bio-ink formulation according to the invention is applied to 3D bioprinting technology to produce the three-dimensional object.

[0098] Similar to thermoplastics commonly used in traditional 3D printing, bio-inks can be extruded into filaments through a printing nozzle or needle, which retain their shape after application. However, bio-inks are sensitive to the processing conditions of conventional 3D printing. Compared to traditional 3D printing materials, bio-inks typically require lower printing temperatures (usually 37°C or lower) and milder curing conditions to avoid compatibility with cells and bioactivity.

[0099] Extrusion-based bioprinting and digital light processing (DLP)-based bioprinting are both 3D bioprinting technologies commonly used to produce three-dimensional objects made from biomaterials. In extrusion-based bioprinting, bio-ink in solution or semi-solution form is loaded into a cartridge, and mechanical force (typically air pressure or a motor-driven piston or screw) pushes the bio-ink through a nozzle to form filaments, which can be deposited layer by layer to produce the desired three-dimensional object. DLP-based bioprinting is also a layer-by-layer process. However, instead of extruding material through a nozzle, an illumination source processes each layer with a still image. This image is projected into a vat of photosensitive liquid to trigger a chemical reaction that solidifies the liquid in the illuminated area. The printed three-dimensional object is obtained by stacking these solidified layers on a build platform.

[0100] Traditional bioprinting techniques involve layer-by-layer deposition of material to form the final structure, but a new method called volumetric bioprinting has recently been reported (see: PN Bernal, P. Delrot, D. Loterie, Y. Li, J. Malda, C. Moser and R. Levato, Volumetric Bioprinting of Complex Living-Tissue Constructswithin Seconds, Adv. Mater.(2019, 31, 1904209). Volumetric bioprinting occurs when bio-ink is placed in liquid cells and selectively irradiated with an energy source. This method causes the irradiated material to actively aggregate and contain the final structure. Volumetric bioprinting using bio-ink can significantly reduce fabrication time. In materials science, this represents a breakthrough in the rapid generation of personalized biomaterials.

[0101] Unlike traditional 3D printing materials such as thermoplastics, which are essentially fixed once printed, bio-inks are dynamic systems due to their high water content and typically amorphous structure. Shape fidelity of the bio-ink must also be considered after filament deposition. Finally, printing pressure and nozzle diameter must be considered to minimize shear stress applied to the bio-ink and any cells within it during printing. Excessive shear force can damage or lyse cells, adversely affecting cell viability.

[0102] Preferred 3D bioprinting technologies for the method of producing three-dimensional objects according to the present invention are selected from DLP-based bioprinting, droplet-based bioprinting, extrusion-based bioprinting, forward transfer bioprinting, inkjet bioprinting, integrated bioprinting, laser-induced bioprinting, stereolithography-based bioprinting, magnetic bioprinting, and volumetric bioprinting. These 3D bioprinting technologies enable precise placement / positioning at predetermined locations within a defined 3D structure.

[0103] In a preferred embodiment of the method for producing three-dimensional objects according to the invention, the bio-ink formulation is subjected to irradiation and / or heat treatment to crosslink the polymer contained in the bio-ink formulation. Such irradiation and / or heat treatment is typically performed after the bio-ink formulation has been applied to 3D bioprinting technology and has been deposited to form a three-dimensional object.

[0104] Preferred irradiation treatments include IR irradiation, VIS irradiation, and / or UV irradiation. Preferred IR radiation is in the range of 600 nm to 1 mm, more preferably in the range of 750 nm to 900 nm, and most preferably in the range of 770 nm to 790 nm. Preferred VIS radiation is in the range of 400 nm to 700 nm, more preferably in the range of 400 nm to 500 nm, and most preferably in the range of 400 nm to 450 nm. Preferred UV radiation is in the range of 100 nm to 400 nm, more preferably in the range of 300 nm to 400 nm, and most preferably in the range of 350 nm to 400 nm. Preferred heat treatment includes exposure to elevated temperatures up to 120°C, preferably up to 100°C, and more preferably up to 60°C. Most preferably, heat treatment includes exposure to elevated temperatures between 30°C and 40°C. Depending on the type of bioink formulation and the polymers contained therein, those skilled in the art can determine suitable irradiation and / or heat treatment conditions.

[0105] In the method for producing three-dimensional objects according to the invention, irradiation and / or heat treatment for crosslinking the polymer can optionally be performed during or after printing, depending on the 3D bioprinting technology used, such as extrusion-based bioprinting or DLP-based bioprinting or any other bioprinting technology described above.

[0106] In a third embodiment of the invention, a three-dimensional object is provided, which can be obtained or acquired by the method for producing three-dimensional objects according to the invention. Preferably, the three-dimensional object is used in tissue engineering, regenerative medicine, cell delivery, drug delivery, drug development, wound dressings, biosensors, cosmetics, hygiene products, medical devices, implantable electronic devices and / or other biomedical applications.

[0107] It should be understood that those skilled in the art are free to combine the above-mentioned preferred, more preferred, even more preferred, and most preferred embodiments of the bio-ink formulation, as well as the relevant embodiments of the present invention.

[0108] definition

[0109] As used herein, the term "synthetic bioink formulation" refers to a bioink formulation that does not contain products of animal, plant, or microbial origin. Microorganisms include, but are not limited to, bacteria, protozoa, algae, and fungi. The synthetic bioink formulation of this invention is formulated from a poly(asparagine) (PAspAm)-derived polymer. Bioinks are materials used to produce engineered / artificial tissues / organs, disease models, and organoids using 3D bioprinting. They must meet certain properties, including rheological, mechanical, biological functional, and biocompatibility properties. Bioinks provide highly reproducible and precise control over the manufactured constructs in an automated manner. These inks are considered one of the most advanced tools for tissue engineering and regenerative medicine (TERM).

[0110] As used herein, the term "polymer" includes, but is not limited to, homopolymers, copolymers such as block, random and alternating copolymers, terpolymers, quaternary copolymers, etc., as well as blends and modifications thereof. Furthermore, unless otherwise expressly limited, the term "polymer" should include all possible configurational isomers of the molecule. These configurations include, but are not limited to, isotactic, syndiotactic, and atactic symmetries. A polymer is a high molecular weight molecule whose structure essentially comprises multiple repetitions of units (i.e., repeating units) derived, either practically or conceptually, from lower molecular weight molecules (i.e., monomers).

[0111] As used in this article, the term “monomer” refers to a molecule that can be polymerized to contribute building units (repeating units) to the basic structure of a polymer or oligomer.

[0112] As used herein, the term "copolymer" generally refers to any polymer derived from more than one type of monomer, wherein the polymer comprises more than one type of corresponding repeating unit. In one embodiment, the copolymer is the reaction product of two or more types of monomers, and therefore comprises two or more types of corresponding repeating units. Preferably, the copolymer comprises two, three, four, five, or six types of repeating units. A copolymer obtained by copolymerizing three types of monomers may also be called a ternary copolymer. A copolymer obtained by copolymerizing four types of monomers may also be called a quaternary copolymer. Copolymers can exist as block, random, and / or alternating copolymers.

[0113] As used herein, the term "block copolymer" refers to a copolymer in which adjacent blocks are compositionally different, i.e., adjacent blocks contain repeating units derived from different types of monomers or repeating units derived from the same type of monomer but with different compositions or sequence distributions of repeating units.

[0114] As used herein, the term "random copolymer" refers to a copolymer in which the probability of finding a given repeating unit at any given site in the chain is independent of the properties of adjacent repeating units. Typically, in random copolymers, the sequence distribution of repeating units follows Bernoullian statistics.

[0115] As used herein, the term "alternating copolymer" refers to a copolymer composed of macromolecules containing two kinds of repeating units in an alternating sequence.

[0116] As used herein, the term "crosslinking agent" refers to a reagent that provides a class of reactive substances capable of activating reactive groups to react with another reactive group in a crosslinking reaction. This enables the crosslinking of two or more polymer chains carrying reactive groups. In the context of this invention, typical crosslinking agents are photoinitiators, thermal initiators, basic initiators, oxidative initiators, enzymes that mediate oxidative crosslinking, and reagents that mediate redox crosslinking. The aforementioned crosslinking agents provide the reactive substances directly, or must be activated for this purpose, for example, through irradiation, heat treatment, etc.

[0117] As used herein, the term "crosslinking additive" refers to a compound capable of reacting with reactive groups in a crosslinking reaction. For example, a crosslinking additive can react with reactive groups in a polymer, thereby providing additional crosslinking.

[0118] In the context of this invention, as used herein, the term "diluent" refers to one or more compounds that act as a solvent, suspending agent, carrier, and / or matrix for polymers and any other components contained in bio-ink formulations. Diluents are typically inert compounds that do not react with said polymers and other components. Typical diluents are compounds that are liquid at room temperature.

[0119] As used herein, the term "shear thinning" refers to the non-Newtonian behavior of a fluid whose viscosity decreases under shear strain. Bioinks with shear-thinning properties enable extrusion at lower extrusion forces, thereby minimizing cell damage during bioprinting. The ratio G'' / G' of the shear loss modulus (G'') to the shear storage modulus (G') in a viscoelastic material is defined as tan δ (the loss tangent), which provides a measure of the material's damping. Tan δ can also be imagined as the tangent of the phase angle δ between the storage modulus and the loss modulus. Shear thinning refers to the decrease in viscosity with increasing shear rate. This is important in extrusion-based printing processes because extruding high-viscosity bioinks at relatively high speeds (high shear rates) induces high shear stress, which can kill cells. If a bioink is shear-thinned—meaning its viscosity decreases during extrusion (high shear rate)—it reduces the shear stress applied to the cells, thereby protecting them from damage. To some extent, shear-thinned bio-inks under extrusion (high shear rate) will become less viscous and more fluid, indicating higher G” and / or lower G’.

[0120] As used herein, the term "supramolecular interaction" refers to a class of molecular interactions classified according to their non-covalent properties. Supramolecular interactions include host-guest interactions or self-assembly interactions based on, for example, van der Waals forces, π-π stacking, hydrogen bonding, hydrophobic interactions, metal-ligand coordination, and / or electrostatic interactions.

[0121] As used herein, the term "alkyl" refers to a saturated hydrocarbon chain, such as, but not limited to, methyl, ethyl, propyl, and butyl. Alkyl groups can be straight-chain or branched. For example, as used herein, propyl includes n-propyl and isopropyl; butyl includes n-butyl, sec-butyl, isobutyl, and tert-butyl, etc. Divalent alkyl groups are also referred to as "alkylene groups" in this application. This nomenclature is familiar to those skilled in the art.

[0122] As used herein, the term "allyl" refers to a substituent having the structural formula R-CH2-CH=CH2, where R is any other group or H. It consists of a methylene bridge (-CH2-) attached to the vinyl group (-CH=CH2).

[0123] As used herein, the term "vinyl" refers to a substituent having the structural formula RC(R')=CH2, wherein R and R' are independently any other radical or H. Preferably, it consists of vinyl (-CH=CH2).

[0124] The invention is further illustrated by the examples described below, but these examples should not be construed as limiting. Those skilled in the art will recognize that various modifications, additions, and substitutions can be made to the invention without departing from the spirit and scope of the invention as defined in the appended claims.

[0125] Example

[0126] Example 1

[0127] Synthesis of methacrylated poly(asparagine) (PAspAm-MA) (1)

[0128] PAspAm-MA (1) was synthesized by ring-opening of polysuccinimide using aminoethyl methacrylate. Polysuccinimide (500 mg) was dissolved in 5 mL of anhydrous dimethylformamide (DMF), and aminoethyl methacrylate hydrochloride (237 mg) was added to this solution. The reaction was carried out at room temperature for 24 hours in the presence of 0.72 mL of triethylamine, and then excess 2-(2-aminoethoxy)ethanol (0.79 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 24 hours. Both steps were carried out under a nitrogen atmosphere. The reaction mixture was first precipitated in diethyl ether, then dissolved in deionized water, followed by dialysis for 2–3 days and lyophilization. 1 The degree of substitution of methacrylate groups on polysuccinimide was determined by ¹H NMR (Bruker 500MHz) using D₂O as a solvent. All spectra were recorded at room temperature.

[0129] pass 1 H NMR confirmed the chemical structure of PAspAm-MA (1) (see [reference]). Figure 1 The methylene (2H) and methine (1H) protons on the poly(asparagine) backbone exhibit peaks at 2.5–2.9 ppm and 4.6 ppm, respectively. The acrylic protons (2H) of methacrylates are located at 5.6 and 6.0 ppm. The peak at 3.3–3.6 ppm indicates the methylene proton (2H) in 2-(2-aminoethoxy)ethanol. 1 The degree of substitution of the methacrylate group calculated by H NMR is 7.2%.

[0130] Rheological properties of PAspAm-MA (1)

[0131] Rheological measurements were performed at 25°C on a Discovery HR-2 hybrid rheometer (TA instruments) equipped with a 20 mm diameter plate-to-plate geometry. PAspAm-MA (1) samples were in-situ photocrosslinked and subjected to an oscillating time scan for 5 minutes at 1% shear strain and a frequency of 1 Hz. Samples were subjected to visible light (OmniCure LX500, 405 nm, 8.8 mW / cm²) after the start of the time scan experiment. 2Irradiation for 60 seconds. Approximately 320 µL of PAspAm-MA (1) solution containing 0.25% wt / v phenyl (2,4,5-trimethylbenzoyl) lithium phosphine (LAP) (5% and 10% wt / v in deionized water) was used for each test and the geometric gap was set to 1 mm.

[0132] Time-scan experiments were conducted using shear storage modulus (G') and shear loss modulus (G'') versus time to determine the gelation behavior during photocrosslinking. Figure 2 As shown, both 5% and 10% PAspAm-MA (1) solutions rapidly form gels, and their shear moduli plateau within 30 and 60 seconds of exposure, respectively. The G' and G'' of the cured PAspAm-MA (1) hydrogels increase significantly with increasing polymer concentration. The 5% PAspAm-MA (1) hydrogel has a storage modulus of 0.1 kPa, while the 10% PAspAm-MA (1) hydrogel has a storage modulus of 3 kPa.

[0133] Cell viability in PAspAm-MA (1) hydrogel

[0134] Mouse 3T3 fibroblasts (ATCC) were cultured at 37°C in Dulbecco modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Corning) at a humidified atmosphere containing 5% CO2. PAspAm-MA (1) polymer and LAP were dissolved in phosphate-buffered saline (PBS) and filtered through a 0.2 µm filter before being mixed with the cell suspension. The final concentrations of each component were 5% and 10% wt / v PAspAm-MA (1) polymer, 0.25% wt / v LAP, and 2.5 M / mL mouse 3T3 fibroblasts, respectively. The polymer / cell mixture solution was added to 96-well plates at 32 μL / well and incubated under 405 nm light (8.8 mW / cm²). 2 Cured for 1 minute. Cell viability in cell-loaded PAspAm-MA (1) hydrogels was quantified on days 1 and 3 (n=4) using the PrestoBlue® Cell Viability Reagent (Invitrogen). Cell growth in PAspAm-MA (1) hydrogels was visualized using a live / dead cell staining kit (live, calcein-AM; dead, propidium iodide; MilliporeSigma).

[0135] Live / dead staining and resazurin assays were used to qualitatively and quantitatively assess cell viability in PAspAm-MA (1) hydrogels cultured for 3 days (see [link]).Figure 3 Live / dead staining images showed that 3T3 fibroblasts in 5% and 10% PAspAm-MA(1) hydrogels were quite viable, and a small fraction of the cells began to elongate on day 3 (see [link to image]). Figure 3 A). The cell survival rate of 3T3 fibroblasts in PAspAm-MA (1) hydrogel was higher than 93% from day 1 to day 3 (see Figure 3 B). PrestoBlue® assays showed that cell metabolism in the 10% PAspAm-MA (1) hydrogel was significantly higher than that in the 5% PAspAm-MA (1) hydrogel during the 3-day culture period (see [link]). Figure 3 (C) This may be because the 5% PAspAm-MA (1) hydrogel network was too loose to retain all the seeded cells, resulting in cell loss into the culture medium. There was no significant difference in cell viability between day 1 and day 3 within each group. These results support the conclusion that PAspAm-MA (1) does not have significant cytotoxicity and therefore has potential for biomedical applications.

[0136] Preparation of PAspAm-MA (1) Bio-ink Formulation

[0137] Bio-ink was prepared by dissolving 500 mg of PAspAm-MA (1) in 5 mL of PBS buffer. LAP (25 mg) and tartrazine (2.5 mg) were added to the solution, and the mixture was vortexed to dissolve completely. The solution was sonicated for 30 minutes to remove air bubbles and then filtered through a 0.45 μm filter.

[0138] PAspAm-MA (1) Printability of Bio-ink Formulation

[0139] The aforementioned bio-ink formulation was printed on a Lumen X+ (Volumetric Bio and Cellink) digital light processing (DLP) bioprinter. The printing was performed at room temperature under near-visible light exposure (405 nm, 20 mW / cm²). 2 The process was carried out. A size of 25 mm (L) was obtained. 13 mm (W) M-shaped prints with a diameter of 0.6 mm (H) (see...) Figure 4 ).

[0140] Example 2

[0141] Synthesis of Tyramine-functionalized polyasparagine (PAspAm-Tyr) (2)

[0142] PAspAm-Tyr (2) was synthesized via ring-opening nucleophilic derivatization of polysuccinimide using tyramine. Polysuccinimide (1,000 mg) was dissolved in 10 mL of anhydrous dimethylformamide (DMF). Tyramine (144.2 mg) was first added to the 10 mL DMF and stirred at room temperature under N2 for 24 hours. Then, excess 2-[2-(2-aminoethoxy)ethoxy]ethanol (2.67 mL) was added to the reaction mixture, and the mixture was stirred at room temperature under N2 for another 24 hours. The reaction mixture was first precipitated in diethyl ether, then dissolved in deionized water, followed by dialysis for 2–3 days and lyophilized.

[0143] pass 1 H NMR confirmed the chemical structure of PAspAm-Tyr (2) (see [reference]). Figure 5 The methylene (2H) and methine (1H) protons on the polysuccinimide backbone exhibit peaks at 2.5–2.9 ppm and 4.6 ppm, respectively. The benzene ring proton (-C6H4-) on the grafted tyramine is located at 6.7 and 7.0 ppm, indicating successful tyramine functionalization. The peak at 3.3–3.7 ppm indicates the methylene proton (2H) in 2-[2-(2-aminoethoxy)ethoxy]ethanol. 1 The degree of substitution of the tyramine group calculated by H NMR is 9.6%.

[0144] Rheological properties of PAspAm-Tyr (2)

[0145] Rheological measurements were performed at 25°C on a Discovery HR-2 hybrid rheometer (TA instruments) equipped with a 20 mm diameter plate-to-plate geometry. PAspAm-Tyr (2) was in-situ photocrosslinked and subjected to an oscillating time scan for 5 minutes at 1% shear strain and a frequency of 1 Hz. Samples were subjected to visible light (OmniCure LX500, 405 nm, 8.8 mW / cm²) after the start of the time scan experiment. 2 Irradiation for 60 seconds. Approximately 320 µL of PAspAm-Tyr (2) solution (10% wt / v in PBS) containing varying amounts of tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate (Ru; 1 mM, 0.5 mM and 0.25 mM) and sodium persulfate (SPS; 20 mM and 10 mM) was used for each experiment with the geometric gap set to 1 mm.

[0146] The rheological properties of polymers with different amounts of co-photoinitiator were evaluated by rheological measurements, and plotted on... Figure 6The increase in the amount of SPS and Ru led to an increase in the shear storage modulus (G') of the PAspAm-Tyr (2) hydrogel. The highest storage modulus (26.6 ± 0.4 kPa) was found in the sample crosslinked with 1 mM Ru and 20 mM SPS (PAspAm-Tyr-1 mM-20 mM), while the lowest storage modulus (9.0 ± 0.1 kPa) was found in the sample containing 0.25 mM Ru and 10 mM SPS (PAspAm-Tyr-0.25 mM-10 mM) (see Table 1). In addition, when the amount of SPS was constant, the time for complete crosslinking of the hydrogel increased with decreasing Ru concentration (see Table 1). PAspAm-Tyr-0.25 mM-20 mM required the longest crosslinking time (73 seconds) to reach the plateau phase. It is worth noting that, when the Ru amount is constant, a higher amount of SPS results in a longer hydrogel crosslinking time. This is likely due to the higher oxidation / reduction efficiency of Ru in the photopolymerization reaction, which leads to a higher storage modulus (see Table 1 and...). Figure 6 ).

[0147] Table 1. Rheological properties of PAspAm-Tyr (2) under visible light exposure

[0148] Preparation of PAspAm-Tyr (2) Bio-ink Formulation

[0149] Various combinations of PAspAm-Tyr (2) bioink solutions were prepared to investigate the effects of Ru (1 mM, 0.5 mM, 0.25 mM), SPS (10 mM and 20 mM), and the light absorber tartrazine (0.0125% wt / v, 0.025% wt / v, 0.05% wt / v) on the gelation kinetics, printability, and post-bioprinting cell viability of these bioinks. The polymer concentration was maintained at 10% wt / v.

[0150] PAspAm-Tyr (2) Printability of Bio-ink Formulation

[0151] PAspAm-Tyr (2) bio-ink was formulated and printed on a Lumen X+ (Volumetric Bio and Cellink) digital light processing (DLP) bioprinter. The printing was performed at room temperature under near-visible light exposure (405 nm, 20 mW / cm²). 2 (This is followed by a series of seemingly unrelated sentences.) 9 mm 0.5 mm and 34 mm 7.2 mm The 0.5mm grid shape and the word "BIOINK" (see...) Figure 7 ).

[0152] Cell printing and cell viability

[0153] Human bone marrow-derived mesenchymal stem cells (hMSCs; ATCCs) were cultured at 37°C in Dulbecco modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin (Corning), and 8 μg / mL basic human fibroblast growth factor (hBFGF, Sigma) under a humidified atmosphere containing 5% CO2. PAspAm-Tyr(2) polymer was dissolved in PBS with co-photoinitiators (Ru and SPS) and a light absorber (tartrazine) and filtered through a 0.2 µm filter before being mixed with the cell suspension. The final concentrations of each component included 10% wt / v PAspAm-Tyr(2) polymer, 1 mM Ru, 10 mM or 20 mM SPS, 0.0125% tartrazine, and 1.5 M / mL hMSCs, respectively. (Print 4) An 8-disc array, each disk measuring 5 mm (diameter) x 1 mm (height). Exposure intensity: 20 mW / cm². 2 Furthermore, the body exposure for each layer (100 μm thick) was 8.25 seconds in the 10 mM SPS assay and 6.25 seconds in the 20 mM SPS assay. The resulting gel discs were cultured in ultra-low adsorption 24-well plates. Cell viability in the bioprinted PAspAm-Tyr(2) hydrogels was quantified using the PrestoBlue® Cell Viability Reagent (Invitrogen) on days 1, 7, and 14 (n = 4). Cell growth in the PAspAm-Tyr(2) hydrogels was visualized using a live / dead cell staining kit (live, calcein-AM; dead, propidium iodide; MilliporeSigma).

[0154] Live / dead staining and PrestoBlue® assays were used to qualitatively and quantitatively assess cell viability in PAspAm-Tyr(2) bioink hydrogels cultured for 14 days (see [link]). Figure 8 Live and dead staining images showed that hMSCs in 10 mM and 20 mM SPS crosslinked hydrogels were quite active and exhibited a tendency to form cell aggregates (see [link]). Figure 8A). PrestoBlue® assays showed that hMSCs proliferated from day 1 to day 14 in both experiments, and cell metabolism was significantly higher in the 20 mM SPS assay than in the 10 mM SPS assay (see [reference]). Figure 8 B). While not wishing to be bound by theory, it is believed that this may be attributed to the higher storage modulus in the 20 mM SPS experiment, which is more conducive to hMSC growth. The cell viability of hMSCs within the bioprinted PAspAm-Tyr (2) hydrogel was consistently above 95% after 14 days of culture (see [link to article]). Figure 8 C). The results support the conclusion that PAspAm-Tyr (2) bioink has no significant cytotoxicity and can support the proliferation of hMSCs in 14-day culture, thus having great potential for biomedical applications.

[0155] Example 3

[0156] Synthesis of PAspAm-Tyr (PAspAm-Tyr-RGD+) (3) conjugated with RGD-containing peptides

[0157] The following diagrams disclose SEQ ID NOS: 9 and 10 in the order of appearance.

[0158]

[0159] PAspAm-Tyr-RGD+ (3) was synthesized by first reacting polysuccinimide with the RGD-containing peptide Ac-Gly-Lys-Gly-Tyr-Gly-Arg-Gly-Asp-Ser-Pro-Gly-NH2 (SEQ ID NO: 9). Polysuccinimide (680 mg) was dissolved in 10 mL of anhydrous dimethylformamide (DMF). First, the RGD-containing peptide (100 mg) in 10 mL of DMF was added and stirred at room temperature for 24 hours. Then, tyramine (107.9 mg) dissolved in 10 mL of DMF was added to the reaction mixture, and the mixture was reacted at room temperature for another 24 hours. The final step was the addition of excess 2-[2-(2-aminoethoxy)ethoxy]ethanol (1.82 mL) and stirring at room temperature for 24 hours. All steps were performed under a N2 atmosphere. The final solution was first precipitated in diethyl ether, then dissolved in deionized water, followed by dialysis for 2–3 days and lyophilized. 1 The degree of substitution of RGD-containing peptides and tyramine groups on the polysuccinimidyl backbone was determined by ¹H NMR (Bruker 500MHz) using D₂O as a solvent. The molar feed ratios of RGD-containing peptides and tyramine to succinimidyl rings in the backbone were 0.78% and 10.3%, respectively.

[0160] (PAspAm-Tyr-RGD+) (3) Preparation of bio-ink formulations

[0161] PAspAm-Tyr-RGD+ (3) bio-ink consisted of 10% wt / v polymer, 1 mM Ru, 20 mM SPS, and 0.0125% tartrazine. PAspAm-Tyr (2) containing 10% wt / v polymer, 1 mM Ru, 20 mM SPS, and 0.0125% tartrazine was used as a comparison. Poly(ethylene glycol) diacrylate (PEGDA; Mn = 6000) was formulated into a bio-ink with a 10% wt / v polymer concentration, 0.5% wt / v photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP), and 0.01% tartrazine as a control.

[0162] Cell printing and cell viability

[0163] Mouse 3T3 fibroblasts (ATCC) were cultured in Dulbecco modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Corning) at 37°C under a humidified atmosphere containing 5% CO2. The bio-ink prepared above was filtered through a 0.2 µm filter and then mixed with the cell suspension. The final cell density was 1 M / mL. (Print 4) An 8-disc array, each disk measuring 5 mm (diameter) x 1 mm (height). Exposure intensity: 20 mW / cm². 2 Furthermore, the body exposure for each layer (100 μm thick) was 8.25 seconds in the PAspAm-Tyr-RGD+ (3) experiment and 6.25 seconds in the PAspAm-Tyr (2) experiment. PEGDA(6k) bioink required a longer body exposure of 20 seconds for each layer. The resulting gel discs were cultured in ultra-low adsorption 24-well plates. Cell viability in the bioprinted PAspAm-Tyr (2) hydrogels was quantified using the PrestoBlue® Cell Viability Reagent (Invitrogen) on days 1, 7, 14, and 21 (n = 4). Cell growth in the PSI-MA hydrogels was visualized using a live / dead cell staining kit (live, calcein-AM; dead, propidium iodide; MilliporeSigma).

[0164] Live / dead staining and PrestoBlue® assays were used to qualitatively and quantitatively assess cell viability in PAspAm-Tyr-RGD+ (3) bio-ink hydrogels cultured for 21 days (see [link]). Figure 9 and 10 Live / dead staining images showed vigorous proliferation of 3T3 fibroblasts in both PAspAm-Tyr-RGD+ (3) and PAspAm-Tyr (2) hydrogels, with very few dead cells (see [link]). Figure 9 Cells in the PAspAm-Tyr-RGD+ (3) hydrogel exhibit an elongated shape and secrete their own extracellular matrix. In contrast, cells in the PAspAm-Tyr (2) hydrogel form numerous cell aggregates. However, a large number of dead cells were observed in the PEGDA (6k) hydrogel (see [link to PEGDA(6k)]). Figure 9 PrestoBlue® assays showed that 3T3 fibroblasts in both groups proliferated rapidly from day 1 to day 21 (see PrestoBlue® assay). Figure 10 A). The metabolic activity of cells in the PAspAm-Tyr-RGD+ (3) bio-ink hydrogel formulation increased 28-fold from day 1 to day 21, while the metabolic activity of cells in the PAspAm-Tyr (2) bio-ink hydrogel formulation increased 17-fold after 21 days of culture. The metabolic activity of the PEGDA(6k) bio-ink hydrogel formulation was close to 0 from day 1, indicating that most cells had already died by day 1 after printing. The cell viability of 3T3 fibroblasts in the bioprinted PAspAm-Tyr-RGD+ (3) and PAspAm-Tyr (2) hydrogels was above 95% after 21 days of culture (see [link]). Figure 10 B). The results confirmed that the PAspAm-Tyr-RGD+ (3) bioink has high cell affinity because the RGD+ peptide acts as a cell binding site that enhances cell attachment and proliferation. Compared to synthetic PEGDA bioinks, in which most 3T3 fibroblasts die on day 1 of culture, our synthetic PAspAm-Tyr-RGD+ (3) and PAspAm-Tyr (2) bioinks are cell-supportive and have great potential for a variety of biomedical applications.

[0165] Example 4

[0166] Synthesis of norbornene-functionalized polyasparagine (PAspAm-Nor) (4)

[0167] PAspAm-Nor (4) was synthesized via a ring-opening nucleophilic reaction of polysuccinimide with 5-norbornene-2-methylamine derivatization (see [link to original text]). Figure 11A). Polysuccinimide (1,000 mg) was dissolved in 10 mL of anhydrous dimethylformamide (DMF). First, 5-norbornene-2-methylamine (414.4 mg) was added to 10 mL of DMF and stirred at room temperature under N2 for 24 hours. Then, 1.48 mL of 2-(2-aminoethoxy)ethanol was added to the reaction mixture and stirred at room temperature under N2 for another 24 hours. The reaction mixture was first precipitated in diethyl ether, then dissolved in deionized water, followed by dialysis for 3 days and lyophilization.

[0168] pass 1 H NMR confirmed the chemical structure of PAspAm-Nor (4) (see [reference]). Figure 11 B). The methylene (2H) and methine (1H) protons on the polysuccinimide backbone exhibit peaks at 2.5–2.9 ppm and 4.6 ppm, respectively. The peaks at 5.9 and 6.1 ppm are attributed to the double bond (CH=CH) on the norbornene group. The peak at 3.3–3.7 ppm indicates the methylene proton (2H) in 2-(2-aminoethoxy)ethanol. 1 The degree of substitution of the norbornene group calculated by H NMR is 32%.

[0169] Preparation of PAspAm-Nor (4) Bio-ink Formulation

[0170] The bio-ink (PAspAm-Nor) composed thereof was formulated by mixing the PAspAm-Nor (4) polymer with a thiol-functionalized crosslinking molecule in water. The crosslinking molecule was 4-arm-PEG-SH (MW = 5 kDa). The total mass concentration of PAspAm-Nor (4) polymer and 4-arm-PEG-SH in water was 10% wt / v. The photoinitiator and photoabsorber consisted of 0.5% wt / v lithium phenyl (2,4,5-trimethylbenzoyl)phosphinate (LAP) and 0.025% tartrazine, respectively.

[0171] When irradiated with 405 nm light under ambient conditions, crosslinking occurs through click chemistry between the PAspAm-Nor (4) polymer and the norbornene and thiol groups of the 4-arm-PEG-SH polymer, respectively.

[0172] Preparation of PAspAm-Nor-RGD++ (4a) Bio-ink Formulation

[0173] To enhance the biofunctionality of PAspAm-Nor (4) bioink, a thiol-containing peptide (RGD++) with the sequence H-Gly-Arg-Gly-Asp-Ser-Pro-Cys-OH (SEQ ID NO: 11) was added to the bioink (i.e., pre-crosslinked PAspAm-Nor (4) bioink) prepared above, with a molar ratio of 10% to norbornene functional groups.

[0174] like Figure 12 As shown in the reaction diagram, when irradiated with 405 nm light under ambient conditions, crosslinking occurs through click chemistry between norbornene and 4-arm-PEG-SH thiol groups of the PAspAm-Nor (4) polymer and thiol groups of RDG-containing peptides.

[0175] Rheological properties of PAspAm-Nor (4) and PAspAm-Nor-RGD++ (4a)

[0176] Rheological measurements were performed at 25 °C on a Discovery HR-2 hybrid rheometer (TA instruments) equipped with a 20 mm diameter plate-to-plate geometry. The bio-inks (4) and (4a) prepared above were in situ photocrosslinked and subjected to an oscillating time-scan at 1% shear strain and 1 Hz for 5 minutes. Samples were subjected to visible light (OmniCure LX500, 405 nm, 8.8 mW / cm²) after the start of the time-scan experiment. 2 Irradiate for 60 seconds. Set the geometric gap to 1 mm.

[0177] Time-scan experiments were conducted using storage modulus (G') and loss modulus (G'') versus time to determine the gelation behavior during photocrosslinking. Figure 13 As shown, both PAspAm-Nor (4) and PAspAm-Nor-RGD++ (4a) bioinks rapidly crosslinked into mechanically strong hydrogels within 2 seconds. The G' of the cured PAspAm-Nor (4) hydrogel was 34.5 k ± 2.1 Pa, while that of the cured PAspAm-Nor-RGD++ (4a) hydrogel was 32.7 ± 1.8 kPa, indicating that the addition of RGD++ peptides with a molar ratio of 10% to norbornene groups had no significant effect on the stiffness of the hydrogels.

[0178] Cell printing and cell viability

[0179] Mouse 3T3 fibroblasts (ATCC) were cultured at 37°C in Dulbecco modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Corning) at a humidified atmosphere containing 5% CO2. PAspAm-Nor (4) and PAspAm-Nor-RGD++ (4a) bioinks were filtered through a 0.2 µm filter and then mixed with cells at a density of 1 M / mL. (Print 4) An array of 8 disks, each disk measuring 5 mm (diameter). 1 mm (height). Exposure intensity: 20 mW / cm 2 The body exposure for each layer (100 μm thick) was 2.25 seconds. The resulting gel discs were cultured in ultra-low adsorption 24-well plates. Cell viability in the bioprinted PAspAm-Nor (4) and PAspAm-Nor-RGD++ (4a) hydrogels was quantified using the PrestoBlue® Cell Viability Reagent (Invitrogen) on days 1, 7, 14, 21, and 28 (n = 4). Cell growth in the PAspAm-Nor (4) and PAspAm-Nor-RGD++ (4a) hydrogels was visualized using a live / dead cell staining kit (live, calcein-AM; dead, propidium iodide; MilliporeSigma).

[0180] Live / dead staining and PrestoBlue® assays were used to qualitatively and quantitatively assess cell viability in PAspAm-Nor(4) and PAspAm-Nor-RGD++(4a) bioink hydrogels cultured for 28 days (see [link]). Figure 14 Live / dead staining images showed that 3T3 fibroblasts proliferated vigorously in both PAspAm-Nor-RGD++ (4a) and PAspAm-Nor (4) hydrogels, with very few dead cells (see [link]). Figure 14 A). Cells in the PAspAm-Nor-RGD++ (4a) hydrogel exhibited an elongated shape and secreted their own extracellular matrix. In contrast, cells in the PAspAm-Nor (4) hydrogel remained round. PrestoBlue® assays showed that 3T3 fibroblasts in both groups proliferated rapidly from day 1 to day 28 (see [link]). Figure 14B). The metabolic activity of the PAspAm-Nor-RGD++ (4a) group increased 28-fold from day 1 to day 28, while the metabolic activity of the PAspAm-Nor (4) group increased 5-fold after 28 days of culture. The results confirm that the PAspAm-Nor-RGD++ (4a) bioink has high bioactivity because the RGD-containing peptides act as cell binding sites that can enhance cell attachment and proliferation.

[0181] The embodiments provided herein are not intended to limit the scope of the invention as set forth in the claims in any way.

Claims

1. A bio-ink formulation comprising a polymer, wherein the polymer comprises repeating unit U1 and repeating unit U1': ， in: R 1 Selected from alkylene groups, preferably from C1-C4 groups. 20 Alkylene, more preferably selected from C1-C 10 Alkylene, most preferably selected from methylene, ethylene, and propylene; R 11 Selected from hydrogen or alkyl groups, preferably selected from hydrogen or C1-C 20 Alkyl groups, more preferably selected from hydrogen or C1-C 10 Alkyl groups, most preferably selected from hydrogen, methyl, ethyl, and propyl; A 1 It is the structural part that imparts hydrophilicity to the polymer; n1 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; and m1 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000.

2. The bio-ink formulation according to claim 1, wherein A 1 It contains one or more groups selected from -OH, -SH, -NH2, -CO-NH2 and -CO2H.

3. The bio-ink formulation according to claim 1 or 2, wherein A 1 Contains an alkylene unit -(CH2) x - and / or ethylene oxide units -(CH2-CH2-O) y - where x and y are independent integers from 1 to 2,000.

4. The bio-ink formulation according to one or more of claims 1 to 3, wherein the polymer further comprises repeating unit U2 and repeating unit U2': ， in: R 2 The components are independently selected from alkylene groups, preferably from C1-C2 groups. 20 Alkylene, more preferably selected from C1-C 10 Alkylene, most preferably selected from methylene, ethylene, and propylene; R 22 The components are independently selected from hydrogen or alkyl groups, preferably from hydrogen or C1-C groups. 20 Alkyl groups, more preferably selected from hydrogen or C1-C 10 Alkyl groups, most preferably selected from hydrogen, methyl, ethyl, and propyl; A 2 It is a structural part containing at least one reactive group that can further react to crosslink two or more polymer chains; n2 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; and m2 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000.

5. The bio-ink formulation according to claim 4, wherein A 2 The reactive groups contained therein are selected from groups capable of undergoing polymerization crosslinking reactions, groups capable of undergoing Michael-type or click-type crosslinking reactions, groups capable of undergoing oxidative crosslinking reactions, and groups capable of undergoing redox crosslinking reactions.

6. The bio-ink formulation according to claim 4 or 5, wherein A 2 The reactive groups contained herein are selected from groups capable of undergoing polymerization and crosslinking reactions, wherein preferably the groups contain one or more vinyl or allyl groups.

7. The bio-ink formulation according to claim 4 or 5, wherein A 2 The reactive groups contained herein are selected from groups capable of undergoing Michael-type or click-chemical crosslinking reactions, wherein preferably the groups contain one or more acrylate, methacrylate, n-alkyne, cycloalkyne, amine, azide, carboxyl, hydrazide, hydroxyl, maleimide, norbornene, tetrazine and / or thiol groups.

8. The bio-ink formulation according to claim 4 or 5, wherein A 2 The reactive groups contained herein are selected from groups capable of undergoing oxidative crosslinking reactions, wherein preferably the groups contain one or more aromatic structural moieties having one or more aromatic hydroxyl groups, wherein more preferably the groups contain one or more monophenol, diphenol, triphenol, oligophenol and / or polyphenol structural moieties, wherein particularly preferably the groups contain monophenol structural moieties.

9. The bio-ink formulation according to claim 4 or 5, wherein A 2 The reactive groups contained herein are selected from groups capable of undergoing redox crosslinking reactions, wherein preferably the groups contain one or more thiol and / or amine groups.

10. The bio-ink formulation according to any one of claims 4 to 9, further comprising the ability to interact with A 2 A reactive peptide compound, wherein preferably the reactive peptide compound comprises one or more components capable of reacting with A. 2 The reactive functional groups contained therein are reactive functional groups, wherein the functional groups are preferably selected from allyl, vinyl, acrylate, methacrylate, maleimide and thiol.

11. The bio-ink formulation according to one or more of claims 1 to 10, wherein the polymer further comprises (i) repeating unit U3 and repeating unit U3'; (ii) repeating unit U4 and repeating unit U4'; or (iii) repeating unit U3, repeating unit U3', repeating unit U4 and repeating unit U4': ， in: R 3 and R 4 The components are independently selected from alkylene groups, preferably from C1-C2 groups. 20 Alkylene, more preferably selected from C1-C 10 Alkylene, most preferably selected from methylene, ethylene, and propylene; R 33 and R 44 The components are independently selected from hydrogen or alkyl groups, preferably from hydrogen or C1-C groups. 20 Alkyl groups, more preferably selected from hydrogen or C1-C 10 Alkyl groups, most preferably selected from hydrogen, methyl, ethyl, and propyl; A 3 It is the structural part that gives the polymer cells their function; A 4 It is the structural part that imparts shear-thinning properties to the polymer; In case (i): n3 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; m3 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; and The sum of n1, n2, n3, m1, m2, and m3 is greater than 5, preferably greater than 50, and more preferably greater than 500; or In case (ii): n4 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; m4 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; and The sum of n1, n2, n4, m1, m2, and m4 is greater than 5, preferably greater than 50, more preferably greater than 500, or In case (iii): n3 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; m3 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; n4 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; m4 is an integer from 1 to 5,000, preferably from 10 to 5,000, and more preferably from 100 to 5,000; and In case (iii), the sum of n1, n2, n3, n4, m1, m2, m3 and m4 is greater than 7, preferably greater than 50, and more preferably greater than 500.

12. The bio-ink formulation according to claim 11, wherein A 3 It contains one or more synthetic functional peptide sequences present in natural proteins, wherein the natural protein is preferably fibronectin, laminin, collagen, elastin, albumin, immunoglobulin or filament.

13. The bio-ink formulation according to claim 11 or 12, wherein A 3 Contains selected from RGD, YIGSR (SEQ ID NO: 1), IKVAV (SEQ ID NO: 2), DGEA (SEQ ID NO: 3), PHSRN (SEQ ID NO: 4), PRARI (SEQ ID NO: 5), (GAGAGS) p (SEQ ID NO: 6), (VPGXG) n (SEQ ID NO: 7), (GPO) m One or more synthetic functional peptide sequences of Gly-Lys-Gly-Tyr-Gly-Arg-Gly-Asp-Ser-Pro-Gly (SEQ ID NO: 8), wherein p, n and m are independent integers ≥ 1.

14. The bio-ink formulation according to one or more of claims 11 to 13, wherein A 4 It is the structural part that acquires shear-thinning properties due to supramolecular interactions.

15. The bio-ink formulation according to claim 14, wherein the supramolecular interaction is a host-guest or self-assembly interaction, wherein such interaction preferably includes the interaction between cyclodextrin, adamantane, cucurbita[8]urea, cholesterol, polyethylene glycol and ureidopyrimidinone.

16. The bio-ink formulation according to claim 14 or 15, wherein A 4 It contains one or more cyclodextrin, adamantane, cucurbita[8]urea, cholesterol, polyethylene glycol or ureidopyrimidinone structural moieties.

17. The bio-ink formulation according to one or more of claims 1 to 16, wherein the polymer has a mass concentration of 0.1% (wt / v) to 99% (wt / v) based on the total volume of the bio-ink formulation, preferably 1% (wt / v) to 70% (wt / v), more preferably 2% (wt / v) to 40% (wt / v), particularly preferably 5% (wt / v) to 30% (wt / v), and most preferably 5% (wt / v) to 20% (wt / v).

18. The bio-ink formulation according to one or more of claims 1 to 17, further comprising one or more selected from crosslinking agents and crosslinking additives.

19. The bio-ink formulation according to claim 18, wherein the crosslinking agent is selected from photoinitiators, thermal initiators, basic initiators, oxidative initiators, enzymes that mediate oxidative crosslinking, and reagents that mediate redox crosslinking.

20. The bio-ink formulation according to claim 18 or 19, wherein the crosslinking additive is a compound comprising two or more polymerizable groups having at least one C=C double bond, wherein preferably, the polymerizable group having at least one C=C double bond is selected from acrylates, methacrylates, allyl, styrene, and vinyl.

21. The bio-ink formulation according to claim 18 or 19, wherein the crosslinking additive is a compound comprising two or more thiol groups, preferably three or more thiol groups, more preferably four thiol groups.

22. A method for producing a three-dimensional object, wherein a bio-ink formulation according to one or more of claims 1 to 21 is applied to a 3D bioprinting technique to produce the three-dimensional object.

23. The method of claim 22, wherein the bio-ink formulation is subjected to irradiation and / or heat treatment to crosslink the polymer contained in the bio-ink formulation.

24. A three-dimensional object which can be obtained by the method according to claim 22 or 23.

25. The three-dimensional object of claim 24, wherein the three-dimensional object is used in tissue engineering, regenerative medicine, cell delivery, drug delivery, drug development, wound dressings, biosensors, cosmetics, hygiene products, medical devices, implantable electronics and / or other biomedical applications.

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